Methods for reducing oxidative damage

ABSTRACT

The invention provides a method for reducing oxidative damage in a mammal, a removed organ, or a cell in need thereof. The method comprises administering an effective amount of an aromatic cationic peptide. The aromatic cationic peptide has (a) at least one net positive charge; (b) a minimum of three amino acids; (c) a maximum of about twenty amino acids, (d) a relationship between the minimum number of net positive charges (p m ) and the total number of amino acid residues (r) wherein 3 p m  is the largest number that is less than or equal to r+1; (e) a relationship between the minimum number of aromatic groups (a) and the total number of net positive charges (p t ) wherein 3a or 2a is the largest number that is less than or equal to p t +1, except that when a is 1, p t  may also be 1; and (f) at least one tyrosine or tryptophan amino acid.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/955,412, filed Dec. 1, 2015, now U.S. Pat. No. 9,950,026, which is acontinuation of U.S. application Ser. No. 14/100,626, filed Dec. 9,2013, now U.S. Pat. No. 9,623,069, which is a continuation of U.S.application Ser. No. 12/843,333, filed Jul. 26, 2010, now U.S. Pat. No.8,618,061, which is a continuation of U.S. application Ser. No.11/428,188, filed Jun. 30, 2006, now U.S. Pat. No. 7,781,405, which is acontinuation application of U.S. application Ser. No. 11/040,242 filedon Jan. 21, 2005, now U.S. Pat. No. 7,550,439, which claims priority toU.S. Provisional Application No. 60/538,841 filed on Jan. 23, 2004, thecontents of which are hereby incorporated by reference in theirentireties.

GOVERNMENT INTEREST

This invention was made with government support from the NationalInstitute on Drug Abuse under Grant No. PO1 DA08924. The U.S. governmenthas certain rights in this invention.

BACKGROUND OF THE INVENTION

Mitochondria are essential to cell survival as the main producers of ATPvia oxidative phosphorylation. However, the mitochondria respiratorchain is also a major source of oxidative free radicals. For example,radical production can occur as a result of the reaction ofmitochondrial electron carriers, such as ubiquinol, with oxygen to forma superoxide. Superoxides react by dismutation to hydrogen peroxide,which can decompose to hydroxyl radical. In addition, superoxides reactwith nitric oxide to form peroxynitrite and other reactive oxidants.

Aging is associated not only with increased reactive oxygen species(ROS) production, but also a decrease in the endogenous antioxidantdefense mechanisms. Mitochondria are particularly vulnerable tooxidative stress because they are continuously exposed to ROS. As aconsequence, mitochondria decay is often associated with aging.

Free radicals, including ROS, and reactive nitrogen species (RNS)produce diverse non-specific damage to biological molecules, includinglipids, proteins, RNA and DNA. Such damage of these molecules has beenimplicated in numerous clinical disorders, such as atherosclerosis,preeclampsia, Alzheimer's disease, Parkinson's disease and arthritis.

Antioxidant therapy can potentially delay the aging process, and bebeneficial in a host of human diseases and conditions such as thosedescribed above. However, the development of specific mitochondrialtherapies has been hampered by the difficulty of delivering antioxidantmolecules to mitochondria in vivo. For example, the molecule must firstbe taken up across the plasma membrane into the cytoplasm, and thentargeted selectively to mitochondria.

None of the currently available antioxidant compounds specificallytarget mitochondria. The endogenous antioxidants, superoxide dismutaseand catalase, are poorly absorbed orally, have short half-lives, and donot cross the blood-brain barrier. The natural antioxidants (e.g.,Vitamin E, coenzyme Q, polyphenols) are not water-soluble and tend toaccumulate in cell membranes and only cross the blood-brain barrierslowly.

Therefore, there is a need for improved methods of reducing oxidativedamage with antioxidative compounds that cross cell membranes. Inaddition, it would also be beneficial for the antioxidative compounds tospecifically target mitochondria.

SUMMARY OF THE INVENTION

These and other objectives have been met by the present invention whichprovide a method for reducing oxidative damage in a mammal in needthereof. The method comprises administering to the mammal an effectiveamount of an aromatic cationic peptide. The aromatic cationic peptidehave (a) at least one net positive charge; (b) a minimum of three aminoacids; (c) a maximum of about twenty amino acids; (d) a relationshipbetween the minimum number of net positive charges (p_(m)) and the totalnumber of amino acid residues (r) wherein 3p_(m) is the largest numberthat is less than or equal to r+1; (e) a relationship between theminimum number of aromatic groups (a) and the total number of netpositive charges (p_(t)) wherein 3a is the largest number that is lessthan or equal to p_(t)+1, except that when a is 1, p_(t) may also be 1;and (f) at least one tyrosine or tryptophan amino acid.

In another embodiment, the invention also provides a method of reducingoxidative damage in a removed organ of a mammal. The method comprisesadministering to the removed organ an effective amount of anaromatic-cationic peptide. The aromatic-cationic peptide have (a) atleast one net positive charge; (b) a minimum of four amino acids; (c) amaximum of about twenty amino acids; (d) a relationship between theminimum number of net positive charges (p_(m)) and the total number ofamino acid residues (r) wherein 3p_(m) is the largest number that isless than or equal to r+1; (e) a relationship between the minimum numberof aromatic groups (a) and the total number of net positive charges(p_(t)) wherein 2a is the largest number that is less than or equal top_(t)+1, except that when a is 1, p_(t) may also be 1; and (f) at leastone tyrosine or tryptophan amino acid.

In a further embodiment, the invention provides a method of reducingoxidative damage in a mammal in need thereof. The method comprisesadministering) to the mammal an effective amount of an aromatic-cationicpeptide. The aromatic-cationic peptide have (a) at least one netpositive charge; (b) a minimum of three amino acids; (c) a maximum ofabout twenty amino acids; (d) a relationship between the minimum numberof net positive charges (p_(m)) and the total number of amino acidresidues (r) wherein 3p_(m) is the largest number that is less than orequal to r+1; (e) a relationship between the minimum number of aromaticgroups (a) and the total number of net positive charges (p_(t)) wherein2a is the largest number that is less than or equal to p_(t)+1, exceptthat when a is 1, p_(t) may also be 1, and (f) at least one tyrosine ortryptophan amino acid.

In yet a further embodiment the invention provides a method of reducingoxidative damage in a removed organ of a mammal. The method comprisesadministering to the removed organ an effective amount of anaromatic-cationic peptide. The aromatic cationic peptide have (a) atleast one net positive charge; (b) a minimum of three amino acids; (c) amaximum of about twenty amino acids; (d) a relationship between theminimum number of net positive charges (p_(m)) and the total number ofamino acid residues (r) wherein 3p_(m) is the largest number that isless than or equal to r+1; (e) a relationship between the minimum numberof aromatic groups (a) and the total number of net positive charges(p_(t)) wherein 3a is the largest number that is less than or equal top_(t)+1, except that when a is 1, p_(t) may also be 1, and (f) at leastone tyrosine or tryptophan amino acid.

In yet another embodiment, the invention provides a method of reducing,oxidative damage in a cell in need thereof. The aromatic cationicpeptide have (a) at least one net positive charge; (b) a minimum ofthree amino acids; (c) a maximum of about twenty amino acids; (d) arelationship between the minimum number of net positive charges (p_(m))and the total number of amino acid residues (r) wherein 3p_(m) is thelargest number that is less than or equal to r+1; (e) a relationshipbetween the minimum number of aromatic groups (a) and the total numberof net positive charges (p_(t)) wherein a is the largest number that isless than or equal to p_(t)+1, except that when a is 1, p_(t) may alsobe 1, and (f) at least one tyrosine or tryptophan amino acid.

In an additional embodiment, the invention provides a method of reducingoxidative damage in a cell in need thereof. The aromatic cationicpeptide have (a) at least one net positive charge; (b) a minimum ofthree amino acids; (c) a maximum of about twenty amino acids; (d) arelationship between the minimum number of net positive charges (p_(m))and the total number of amino acid residues (r) wherein 3p_(m) is thelargest number that is less than or equal to r+1; (e) a relationshipbetween the minimum number of aromatic groups (a) and the total numberof net positive charges (p_(t)) wherein 2a is the largest number that isless than or equal to p_(t)+1, except that when a is 1, p_(t) may alsobe 1, and (f) at least one tyrosine or tryptophan amino acid

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1B. (A)SS-02 and (B)SS-05 dose-dependently scavenge H₂O₂.

FIGS. 2A-2B. (A)SS-02 dose-dependently inhibits linoleic acidperoxidation induced by ABAP and (B)SS-02 SS-05, SS-29, SS-30, SS-31,SS-32 and Dmt reduced the rate of linoleic acid peroxidation induced byABAP.

FIGS. 3A-3B. (A)SS-02 dose-dependently inhibits LDL oxidation induced by10 mM CuSO₄ and (B)SS-02, SS-05, SS-29, SS-30, SS-31, SS-32 and Dmtreduced rate of LDL oxidation.

FIGS. 4A-4B. (A)SS-02 inhibits mitochondrial production of hydrogenperoxide as measured by luminol chemiluminescence under basal conditionsand upon stimulation by antimycin. (B)SS-02, SS-29, SS-30 and SS-31reduced spontaneous generation of hydrogen peroxide generated byisolated mitochondria.

FIGS. 5A-5B. (A)SS-31 inhibits spontaneous production of hydrogenhydroperoxide by isolated mitochondria and (B)SS-31 inhibits hydrogenperoxide production stimulated by antimycin.

FIGS. 6A-6C. SS-31 dose-dependently decreased intracellular ROS(reactive oxygen species) (A) and increased cell survival (B) in N₂Acells exposed to a high dose of the pro-oxidant t-butyl hydroperoxide(t-BHP; 0.5 mM), (C)SS-02 also dose-dependently increased cell survivalwhen N2A cells were exposed to 1 mM t-BHP.

FIGS. 7A-7B. SS-31 dose-dependently prevented loss of cell viabilitycaused by low doses of t-BHP (0.05-0.1 mM) in neuronal (A) SH-SY5Y and(B) N₂A cells.

FIG. 8. SS-31 dose-dependently decreased the percent of cells showingincreased caspase activity after treatment with a low dose of t-BHP for12 h in N₂A cells.

FIG. 9. SS-31 dose-dependently reduced the rate of ROS accumulation inN₂A cells with 0.1 mM t-BHP over a 4 h period.

FIGS. 10A-10C. SS-31 inhibited lipid peroxidation caused by exposure ofN₂A cells to 1 mM t-BHP for 1 h. (A) untreated cells; (B) cells treatedwith 1 mM t-BHP for 3 h; (C) cells treated with 1 mM t-BHP and 10 nMSS-31 for 3 h.

FIG. 11. SS-31 prevented mitochondrial depolarization and ROSaccumulation in N₂A cells exposed to t-BHP.

FIGS. 12A-12D. SS-31 prevents apoptosis induced by a low dose of t-BHP.Apoptosis was evaluated by confocal microscopy with the fluorescentprobe Hoechst 33342. (FIG. 12A) a representative field of cells nottreated with t-BHP. (FIG. 12AA) Fluorescent image showing a few cellswith dense, fragmented chromatin indicative of apoptotic nuclei. (FIG.12B) A representative field of cells treated with 0.025 mM t-BHP for 24h. (FIG. 12BB) Fluorescent image showing an increased number of cellswith apoptotic nuclei. (FIG. 12C) A representative field of cellstreated with 0.025 mM t-BHP and 1 nM SS-31 for 24 h. (FIG. 12CC)Fluorescent image showing a reduced number of cells with apoptoticnuclei. (FIG. 12D)SS-31 dose-dependently reduced the percent ofapoptotic cells caused by 24 h treatment with a low dose of T-BHP (0.05mM).

FIG. 13A(a-e). SS-02 and SS-31 reduced lipid peroxidation in isolatedguinea pig hearts subjected to warm reperfusion after a brief period ofischemia. Immunohistochemical analysis of 4-hydroxy-2-nonenol(HNE)-modified proteins in paraffin sections from guinea pig heartsaerobically perfused 30 min with (a) buffer; (b) 100 nM SS-02; (c) 100nM SS-20 and (d) 1 nM SS-31, then subjected to 30 min ischemia andreperfused for 90 min with corresponding peptides. Tissue slices wereincubated with anti-HNE antibody. (e) Background control: stainingwithout primary antibody.

FIG. 13B(a-e). SS-02 and SS-31 reduced lipid peroxidation in isolatedguinea pig hearts subjected to warm reperfusion after a brief period ofischemia. Immunohistochemical analysis of 4-hydroxynonenol(HNE)-modified proteins in paraffin sections from guinea pig heartsaerobically perfused 30 min with buffer, then subjected to 30 minischemia and reperfused with (a) buffer; (b) 100 nM SS-02; (c) 100 nMSS-20 and (d) 1 nM SS-31 for 90 min. Tissue slices were incubated withanti-HNE antibody. (e) Background control: staining without primaryantibody.

FIG. 14A. SS-31 significantly improved coronary flow in isolated guineapig hearts subjected to warm reperfusion after prolonged (18 h) coldischemia. The shaded area represents 18 h of ischemia at 4° C.

FIG. 14B(a-c). Guinea pig hearts perfused with a cardioplegic solution(St. Thomas solution) without (a) or with (b) 1 nM SS-31 for 3 min andthen subjected to 18 h of cold ischemia (4° C.), (c) background stainingwith primary antibody. The hearts were then reperfused with buffer at34° C. for 90 min.

FIG. 14C. SS-31 prevents apoptosis in endothelial cells and myocytes inisolated guinea pig hearts subjected to warm reperfusion after prolonged(18 h) cold ischemia. Guinea pig hearts perfused with a cardioplegicsolution (St. Thomas solution) without or with nM SS-31 for 3 min andthen subjected to 18 h of cold ischemia (4° C.). The hearts were thenreperused with buffer at 34° C. for 90 min. Apoptosis was assessed bythe TUNEL stain (green) and nuclei are visualized by DAPI (blue).

FIG. 15A. SS-31 improves survival of islet cells isolated from mousepancreas as measured by mitochondrial potential. SS-31 (nM) was added toall isolation buffers used throughout the isolation procedure.Mitochondrial potential was measured using TMRM (red) with confocalmicroscopy.

FIGS. 15B & 15C. SS-31 reduces apoptosis and increases viability inislet cells isolated from mouse pancreas as measured by flow cytometry.SS-31 (1 nM) was added to all isolation buffers used throughout theisolation procedure. Apoptosis was ascertained using annexin V andnecrosis by propidium iodide (PI).

FIGS. 16A-16C. SS-31 reduces oxidative damage in pancreatic islet cellscaused by t-butylhydroperoxide (tBHP). Mouse pancreatic islet cells wereuntreated (a), or treated with 25 μM tBHP without (b) or with 1 nM SS-31(c). Mitochondrial potential was measured by TMRM (red) and reactiveoxygen species were measured by DCF (green) using confocal microscopy.

FIG. 17A. SS-31 protects dopamine cells against MPP* toxicity. SN-4741cells were treated with buffer, 50 μM MPP* or 50 μM MPP* and 1 nM SS-31,for 48 h, and the incidence of apoptosis was determined by fluorescentmicroscopy with Hoechst 33342. The number of condensed fragmented nucleiwas significantly increased by MPP* treatment. Concurrent treatment withSS-31 reduced the number of apoptotic cells.

FIG. 17B & FIG. 17BB. SS-31 dose-dependently prevented loss of dopamineneurons in mice treated with MPTP. Three doses of MPTP (10 mg/kg) wasgiven to mice (n=12) 2 h apart. SS-31 was administered 30 min beforeeach MPTP injection, and at 1 h and 12 h after the last MPTP injection.Animals were sacrificed one week later and striatal brain reactions wereimmunostained for tyrosine hydroxylase activity (shown in black).

FIGS. 17C-17E. SS-31 dose-dependently increased striatal dopamine, DOPAC(3,4-dihydroxyphenylacetic acid) and HVA (homovanillic acid) levels inmice treated with MPTP. Three doses of MPTP (10 mg/kg) was given to mice(n=12) 2 h apart. SS-31 was administered 30 min before each MPTPinjection, and at 1 h and 12 h after the last injection. Animals weresacrificed one week later and dopamine, DOPAC and HVA levels werequantified by high pressure liquid chromatography.

FIGS. 18A-18B. SS-31 reduced tBHP-induced LDH release in SH-SY5Y (A) andN₂A (B) cells. Cells were treated with 100 μM tBHP alone, or with SS-31,for 24 h. *P<0.05, **P<0.01, ***P<0.001, compared to tBHP alone.

FIGS. 19A-19C. SS-31 reduced tBHP-induced apoptosis as demonstrated byphosphatidylserine translocation. N₂A cells were incubated with 50 μMtBHP for 6 h and stained with Annexin V and propidium iodide (PI). (A)Untreated cells showed little Annexin V stain and no PI stain. (B) Cellstreated with tBHP showed intense Annexin V staining (green) in mostcells. Combined staining with Annexin V and PI (red) indicate lateapoptotic cells. (C) Concurrent treatment with 1 nM SS-31 resulted in areduction in Annexin V− positive cells and no PI staining.

FIGS. 20A-20C. SS-31 reduced tBHP-induced apoptosis as demonstrated bynuclear condensation. (A)(a-c; a′-c′) N₂A cells were treated with 50 JAMtBHP alone or with SS-31 for 12 h. Cells were stained with Hoechst 33342for 20 min., fixed, and imaged by fluorescent microscopy. (a) Untreatedcells show uniformly stained nuclei (a′). (b) Cells treated with tBHPwere smaller and showed nuclear fragmentation and condensation (b′). (c)Cells treated with tBHP and 1 nM SS-31 had less nuclear changes (c′).(B)-(C)SS-31 dose-dependently reduced percent of apoptotic cells in N₂Acells. Apoptotic cells were counted using MetaMorph software. *P<0.01compared to untreated cells; *P<0.01 compared to tBHP alone. (SS)SS-31dose-dependently reduced percent of apoptotic cells in SH-SY5Y cells.SH-SY5Y cells were treated with 25 μM tBHP for 24 h. *P<0.01 compared tountreated cells; *P<0.01 compared to tBHP alone.

FIGS. 21A-B. SS-31 prevented caspase activation in N₂A cells treatedwith tBHP. (A) incubation of N₂A cells with 100 μM tBHP for 24 hresulted in a significant increase in pancaspase activity that wasdose-dependently prevented by co-incubation with SS-31 (*P<0.01 comparedto tBHP alone). (BXa-c) N₂A cells were treated with 50 μM tBHP for 12 hand stained with caspase-9 FLICA™ kit containing red fluorescentinhibitor SR-LEHD-FMK and Hoechst 33342. (Panel a) Untreated cellsshowed no caspase-9 stain and uniformly stained nuclei. (Panel b) cellstreated with tBHP showed intense caspase-9 activity (red) in cells thatalso show condensed nuclei. (Panel c) Cells treated with tBHP and 1 nMSS-31 showed fewer caspase-9 positive cells and fewer condensed nuclei.

FIGS. 22A-22C. SS-31 dose-dependently reduced intracellular ROSproduction in N₂A cells treated with tBHP. (A) N₂A cells were loadedwith DCFDA, and then exposed to 100 μM tBHP alone, or with SS-31.Intracellular ROS was quantified by the formation of fluorescent DCF.Results shown are mean values (n=3). (BXa-c) N₂A cells were plated inglass bottom dishes and treated with 50 μM tBHP, alone or with 1 nMSS-31, for 6 h. Cells were loaded with DCFDA (10 μM) and imaged byconfocal laser scanning microscopy using ex/em of 495/525 nm. (C) Effectof 1 nM SS-31 in reducing intracellular ROS induced by 50 μM tBHP(*P<0.001 compared to untreated cells; *P<0.05 compared to tBHP alone).

FIGS. 23A-23B. SS-31 protected against tBHP-induced mitochondrialviability. (A)SS-31 protected mitochondrial viability in N₂A cellstreated with tBHP for 24 h. Mitochondrial viability was evaluated usingthe MTT assay (*P<0.01 compared to untreated cells, *P<0.05, P<0.01compared to tBHP alone). (B)SS-31 protected mitochondrial viability inSH-SY5Y cells treated with tBHP for 25 h (*P<0.01 compared to untreatedcells; **P<0.01 compared to tBHP alone).

FIG. 24. Increased hydrogen peroxide (H₂O₂) sensitivity of G93A-SODtransfected murine neuroblastoma (N₂A) cells as compared to wildtypeSOD-transfected N₂A cells after addition of 0.5 or 1 mM H₂O₂ for 1 h.Cell death was quantified by measurement of the percentage ofH₂O₂-induced LDH release of total cellular LDH-content. H₂O₂-induced LDHrelease was significantly reduced by treatment of the cells with 1 to100 μM SS-31 after incubation with H₂O₂. Values are means+S.D., n=4-5,*p<0.1, **p<0.05, Student's t-test.

Black columns wildtype-SOD1-transfected cells, grey columns: G93A-SOD1transfected N₂A cells

FIGS. 25A-25B. (A) Cumulative probability of disease on set and survivalwith SS-31 5 mg/kg/day treatment (n=14) started at symptom onset ascompared to vehicle treatment (n=14). Survival was significantlyimproved by SS-31 (p<0.05, Mantel-Cox log-rank test). (B) Mean survival(days) of G93A mice treated with vehicle or SS-31 5 mg/kg/day. (Data aremean±SD, p<0.05, Student's t-test).

FIG. 26. Effect of SS-31 mg/kg/day on motor performance (seconds) testedby rotarod (Values are mean±standard error of means of the mice stillalive at the respective time point): it was significantly improvedbetween day (d) 110 and day 130 in SS-31-treated animals as compared tothe vehicle-treated group (p<0.005, Repeated Measures ANOVA followed byFisher's PLSD).

FIGS. 27A & 27B. Attenuation of motor neuron loss by SS-31 in theventral horn of the lumbar spinal cord of G93A mice. Photomicrographsshow cresyl violet stained sections through the ventral horn of thelumbar spinal cord from non-transgenic control (A) and G93A mice treatedwith vehicle (PBS) (B) or SS-31 (C) at 110 days of age. Stereologicalanalysis revealed significantly reduced numbers of surviving neurons inG93A mice treated with vehicle as compared to non-transgenic controls(***, p<0.001). This cell loss was significantly ameliorated bytreatment with SS-31 (**, p<0.01). Values are mean±standard error ofmeans.

Differences among means were analyzed using ANOVA followed byNewman-Keuls post hoc test.

FIGS. 28A-28C. 4-hydroxynonenol immunostaining. Photomicrographs ofrepresentative sections through the ventral horn of the lumbar spinalcord of wild-type control (A), and G93A mice treated with vehicle (B) orSS-31 (C) show generalized reduction of 4-hydroxynonenal staining inneurons and neurophils in drug-treated mice.

FIGS. 29A-29C. Nitrotyrosine immunostaining. Photomicrographs ofrepresentative sections through the ventral horn of the lumbar spinalcord of wild-type control (A), and G93A mice treated with vehicle (B) orSS-31 (C) show generalized reduction of nitrotyrosine staining inneurons and neurophils in drug-treated mice.

FIGS. 30A-30F. Temporal changes of cysteine (A, B), ascorbate (C, D) andGSH (E,[[H]]F) levels in post-ischemic brain. C57BU6 mice were subjectedto 30 min MCAO. Values are expressed as nmol/mg protein in cortex (A, C,E) and striatum (B, D, F). Error bars indicate SEM (n=4 animals pergroup). #<0.05 vs 0 h Contral, *p<0.05 vs corresponding Contral, one-wayANOVA with post hoc Fisher's PLSD test. Contral, contralateral side;Ipsil, ipsilateral side; 0 h, sham non-ischemic animal.

FIGS. 31A-31C. Effect of SS-31 peptide on ischemia-induced changes incysteine (A) ascorbate (B), and GSH (C) levels, C57BU6 mice weresubjected to 30 min MCAO and treated with vehicle, SS-31 (2 mg/kg bodyweight) or SS-20 (2 mg/kg body eight) peptide immediately afterreperfusion. Mice were sacrificed at 6 h postischemia. Values areexpressed as percent increase (cysteine) or percent depletion (ascorbateand GSH) in ipsilateral side versus contralateral side. Error barsindicate SEM (n=4-6 animals per group). Note that a difference wasobserved in percent GSH depletion SS-31-treated cortex. *p<0.05 vsvehicle treated group (Veh), one-way ANOVA with post hoc Fisher's PLSDtest.

FIGS. 32A-32F. Effect of SS-31 peptide on ischemia-induced infarct sizeand swelling in C57BU6 mice. Shown are representative serial sections(1.2 mm apart) stained with Cresyl Violet from mice subjected to 30 min(A) and 20 min (B) MCAO and treated with vehicle (Veh) or SS-31 (2 mg/kgbody weight) immediately after reperfusion, 6 h, 24 h, and 48 h. Infarctvolumes ((C) and swelling (D) were estimated at 72 h postischemia from12 serial sections (600 Bm apart) per animal. Mean % cerebral blood flow(CBF) reduction during MCAO (E) and % reperfusion at 10 min postischemia(F) shows no difference between two groups. Error bars indicate SEM (n=1animals per group). *p<0.05 from vehicle treated group (Veh), one-wayANOVA with post hoc Fisher's PLSD test.

FIGS. 33A-33B. SS peptides penetrate islet cells, co-localize withmitochondria and preserve islet mitochondrial membrane potential. (A)Islet cell uptake of SS-31. DBA/2 islet cells were incubated with 1 nMof tritium labeled SS-31 and 1 μM unlabeled SS-31 at 37° C. for 1 h.Following incubation, radioactivity was measured in the medium and incell lysates and the radioactive counts in the medium were subtractedfrom radioactive counts in the cell lysate and normalized to proteincontent. In four consecutive experiments, the mean±(SE) [³H]SS-31 uptakewas 70.2+/31 10.3 pmol/mg, of proteins (B)(i-iv)SS-31 preserves isletmitochondrial potential. DBA/2 mice were treated with SS-31 (3 mg/kgs.c. BID) or vehicle control 24 hours prior to pancreas harvest forislet isolation. SS-31 treated groups had 1 nM SS-31 added to the isletisolation reagents. Following isolation, TMRM uptake was evaluated usingconfocal laser scanning microscopy. Fluorescent (i) and phase (ii)images of TMRM uptake in control mice demonstrate reduced fluorescentuptake indicating mitochondrial depolarization. In sharp contrast,fluorescent (iii) and phase (iv) images of TMRM uptake in SS-31 treatedmice demonstrate increased uptake and preserved mitochondrial potentialindicative of SS-31 protective effect.

FIGS. 34A-34D. SS-31 reduces islet cell apoptosis. DBA/2 mice werepre-treated (24 and 12-hours hours prior to pancreas harvest and isletisolation) with SS-31 (3 mg/kg, s.c., BID) or vehicle control. SS-31 (1nM was added to reagents used for the isolation of islets fromSS-31-treated mice. The islets were dissociated in to single cells withtrypsin/EDTA and were stained with Annexin V-FITC (AnV) and propidiumiodide (PI) and analyzed with the use of dual parameter low cytometry.(A) Percentage of cell undergoing early apoptosis (AnV+cells); P=0.03.(B) Percentage of cells undergoing late apoptosis/early necrosis(AnV+/PI+cells); P=0.03. (C) Percentage of necrotic cells (PI+cells);P=1.0 (D) Percentage of viable cells (AnV−/PI-cells); P=0.03. Data fromindividual pancreatic islet isolations and mean±SE are shown, N=numberof separate islet isolations. Two-tailed P-values were calculated usingMann-Whitney t-test.

FIGS. 35A-35C. Reversal of diabetes following transplantation of amarginal mass of syngeneic islets. Diabetic DBA/2 mice received 200syngeneic islet cells under the right kidney capsule. Reversal ofdiabetes was defined as random nonfasting blood glucose levels below 200mg/dl on 3-consecutive days. (A) Blood glucose levels of each individualcontrol mouse following transplantation of 200 syngeneic islets. (B)Blood glucose levels of each individual SS-31 treated mouse followingtransplantation of 200 syngeneic islets. (C) Reversal of diabetesfollowing transplantation of a marginal islet cell mass in SS-31treatment vs. control. Number of normoglycemic mice by day 1, 3, 5, 10and 14 post transplantation and two-tailed P-valued calculated usingchi-squared bivariate analysis are shown.

DETAILED DESCRIPTION OF THE INVENTION

The invention is based on the surprising discovery by the inventors thatcertain aromatic-cationic peptides reduce oxidative damage. Reducingoxidative damage is important since free radicals, such as ROS and RNS,produce diverse non-specific damage to lipids, proteins. RNA, and DNAoxidative damage caused by free radicals is associated with severaldiseases and conditions in mammals.

Peptides

The aromatic-cationic peptides useful in the present invention arewater-soluble and highly polar. Despite these properties, the peptidescan readily penetrate cell membranes.

The aromatic-cationic peptides useful in the present invention include aminimum of three amino acids, and preferably include a minimum of fouramino acids, covalently joined by peptide bonds.

The maximum number of amino acids present in the aromatic-cationicpeptides of the present invention is about twenty amino acids covalentlyjoined by peptide bonds. Preferably, the maximum number of amino acidsis about twelve, more preferably about nine, and most preferably aboutsix. Optimally, the number of amino acids present in the peptides isfour.

The amino acids of the aromatic-cationic peptides useful in the presentinvention can be any amino acid. As used herein the term “amino acid” isused to refer to any organic molecule that contains at least one aminogroup and at least one carboxyl group. Preferably, at least one aminogroup is at the α position relative to the carboxyl group.

The amino acids may be naturally occurring. Naturally occurring aminoacids include, for example, the twenty most common levorotatory (L)amino acids normally found in mammalian proteins, i.e., alanine (Ala),arginine (Arg), asparagine (Asn) aspartic acid (Asp), cysteine (Cys),glutamine (Glu), glutamic acid (Glu), glycine (Gly), histidine (His),isoleucine (Ileu), leucine (Leu), lysine (Lys), methionine (Met),phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr),trypotophan, (Trp), tyrosine (Tyr), and valine (Val).

Other naturally occurring amino acids include, for example, amino acidsthat are synthesized in metabolic processes not associated with proteinsynthesis. For example, the amino acids ornithine and citrulline aresynthesized in mammalian metabolism during the production of urea.

The peptides useful in the present invention can contain one or morenon-naturally occurring amino acids. The non-naturally occurring aminoacids may be L-, dextrorotatory (D), or mixtures thereof. Optimally, thepeptide has no amino acids that are naturally occurring.

Non-naturally occurring amino acids are those amino acids that typicallyare not synthesized in normal metabolic processes in living organisms,and do not naturally occur in proteins. In addition, the non-naturallyoccurring amino acids useful in the present invention preferably arealso not recognized by common proteases.

The non-naturally occurring amino acid can be present at any position inthe peptide. For example, the non-naturally occurring amino acid can beat the N-terminus, the C-terminus, or at any position between theN-terminus and the C-terminus.

The non-natural amino acids may, for example, comprise alkyl, aryl, oralkylaryl groups. Some examples of alkyl amino acids includeα-aminobutyric acid, β-aminobutyric acid, γ-aminobutyric acid,A-aminovaleric acid, and ε-aminocaproic acid. Some examples of arylamino acids include ortho-, meta, and para-aminobenzoic acid. Someexamples of alkylaryl amino acids include ortho-, meta-, andpara-aminophenyleacetic acid, and γ-phenyl-β-aminobutyric acid.

Non-naturally occurring amino acids also include derivatives ofnaturally occurring amino acids. The derivatives of naturally occurringamino acids may, for example, include the addition of one or morechemical groups to the naturally occurring amino acid.

For example, one or more chemical groups can be added to one or more ofthe 2′, 3′, 4′, 5′, or 6′ position of the aromatic ring of aphenylalanine or tyrosine residue, or the 4′, 5′, 6′ or 7′ position ofthe benzo ring of a tryptophan residue. The group can be any chemicalgroup that can be added to an aromatic ring. Some examples of suchgroups include branched or unbranched C₁-C₄ alkyl, such as methyl,ethyl, n-propyl, isopropyl, butyl, isobutyl, or t-butyl, C₁-C₄ alkyloxy(i.e., alkoxy), amino, C₁-C₄ alkylamino and C₁-C₄ dialkylamino (e.g.,methylamino dimethylamino), nitro, hydroxyl, halo (i.e., fluoro, chloro,bromio, or iodo). Some specific examples of non-naturally occurringderivatives of naturally occurring amino acids include norvaline (Nva),norleucine (Nle), and hydroxyproline (Hyp).

Another example of a modification of an amino acid in a peptide usefulin the methods of the present invention is the derivatization of acarboxyl group of an aspartic acid or a glutamic acid residue of thepeptide. One example of derivatization is amidation with ammonia or witha primary or secondary amine, e.g. methylamine, ethylamine,dimethylamine or diethylamine. Another example of derivatizationincludes esterification with, for example, methyl or ethyl alcohol.

Another such modification includes derivatization of an amino group of alysine, arginine, or histidine residue. For example, such amino groupscan be acylated. Some suitable acyl groups include, for example, abenzoyl group or an alkanoyl group comprising any of the C₁-C₄ alkylgroups mentioned above, such as an acetyl or propionyl group.

The non-naturally occurring amino acids are preferably resistant, andmore preferably insensitive, to common proteases. Examples ofnon-naturally occurring amino acids that are resistant or insensitive toproteases include the dextrorotatory (D-) form of any of theabove-mentioned naturally occurring L-amino acids, as well as L- and/orD-naturally occurring amino acids. The D-amino acids do normally occurin proteins although they are found in certain peptide antibiotics thatare synthesized by means other than the normal ribosomal proteinsynthetic machinery of the cell. As used herein, the 1-amino acids areconsidered to be non-naturally occurring amino acids.

In order to minimize protease sensitivity, the peptides useful in themethods of the invention should have less than five, preferably lessthan four, more preferably less than three, and most preferably, lessthan two contiguous L-amino acids recognized by common proteases,irrespective of whether the amino acids are naturally or non-naturallyoccurring. Optimally, the peptide has only D-amino acids, and no L-aminoacids.

If the peptide contains protease sensitive sequences of amino acids, atleast one of the amino acids is preferably a non-naturally-occurringD-amino acid, thereby conferring protease resistance. An example of aprotease sensitive sequence includes two or more contiguous basic aminoacids that are readily cleaved by common proteases, such asendopeptidases and trypsin. Examples of basic amino acids includearginine, lysine and histidine.

It is important that at least one of the amino acids present in thearomatic-cationic peptide is a tyrosine or tryptophan residue, or aderivative thereof.

It is also important that the aromatic-cationic peptides have a minimumnumber of net positive charges at physiological pH in comparison to thetotal number of amino acid residues in the peptide. The minimum numberof net positive charges at physiological pH will be referred to below as(p_(m)). The total number of amino acid residues in the peptide will bereferred to below as (r).

The minimum number of net positive charges discussed below are all atphysiological pH. The term “physiological pH” as used herein refers tothe normal pH in the cells of the tissues and organs of the mammalianbody. For instance, the physiological pH of a human is normallyapproximately 7.4, but normal physiological pH in mammals may be any pHfrom about 7.0 to about 7.8.

“Net charge” as used herein refers to the balance of the number ofpositive charges and the number of negative charges carried by the aminoacids present in the peptide. In this specification, it is understoodthat net charges are measured at physiological pH. The naturallyoccurring amino acids that are positively charged at physiological pHinclude L-lysine, L-arginine, L-histidine. The naturally occurring aminoacids that are negatively charged at physiological pH include L-asparticacid and L-glutamic acid.

Typically, a peptide has a positively charged N-terminal amino group anda negatively charged C-terminal carboxyl group. The charges cancel eachother out at physiological pH.

In one embodiment of the present invention, the aromatic-cationicpeptides have a relationship between the minimum number of net positivecharges at physiological pH (p_(m)) and the total number of amino acidresidues (r) wherein 3p_(m) is the largest number that is less than orequal to r+1. In this embodiment the relationship between the minimumnumber of net positive charges (p_(m)) ad the total number of amino acidresidues (r) is as follows:

(r) 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 (p_(m)) 1 1 2 2 2 3 33 4 4 4 5 5 5 6 6 6 7

In another embodiment, the aromatic-cationic peptides have arelationship between the minimum number of net positive charges (p_(m))and the total number of amino acid residues (r) wherein 2p_(m) is thelargest number that is less than or equal to r+1. In this embodiment,the relationship between the minimum number of net positive charges(p_(m)) and the total number of amino acid residues (r) is as follows:

(r) 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 (p_(m)) 2 2 3 3 4 4 55 6 6 7 7 8 8 9 9 10 10

In one embodiment, the minimum number of net positive charges (p_(m))and the total number of amino acid residues (r) are equal. In anotherembodiment, the peptides have three or four amino acid residues and aminimum of one net positive charge, preferably, a minimum of two netpositive charges and more preferably a minimum of three net positivecharges.

It is also important that the aromatic-cationic peptides have a minimumnumber of aromatic groups in comparison to the total number of netpositive charges (p_(t)). The minimum number of aromatic groups will bereferred to below as (a).

Naturally occurring amino acids that have an aromatic group include theamino acids histidine, tryptophan, tyrosine, and phenylalanine. Forexample, the hexapeptide Lys-Gln-Tyr-Arg-Phe-Trp has a net positivecharge of two (contributed by the lysine and arginine residues) andthree aromatic groups (contributed by tyrosine, phenylalanine andtryptophan residues).

In one embodiment of the present invention, the aromatic-cationicpeptides useful in the methods of the present invention have arelationship between the minimum number of aromatic groups (a) and thetotal number of net positive charges at physiological pH (p_(t)) wherein3a is the largest number that is less than or equal to p_(t)+1, exceptthat when p_(t) is 1, a may also be 1. In this embodiment, therelationship between the minimum number of aromatic groups (a) and thetotal number of net positive charges (p_(t)) is as follows:

(p+) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 (a) 1 1 1 1 2 22 3 3 3 4 4 4 5 5 5 6 6 6 7

In another embodiment, the aromatic-cationic peptides have arelationship between the minimum number of aromatic groups (a) and thetotal number of net positive charges (p_(t)) wherein 2a is the largestnumber that is less than or equal to p_(t)+1. In this embodiment, therelationship between the minimum number of aromatic amino acid residues(a) and the total number of net positive charges (p_(t)) is as follows:

(p+) 1 2 3 4 5 6 7 8 9 10 1 1 12 13 14 15 16 17 18 19 20 (a) 1 1 2 2 3 34 4 5 5 6 6 7 7 8 8 9 9 10 10

In another embodiment, the number of aromatic groups (a) and the totalnumber of net positive charges (p_(t)) are equal.

Carboxyl groups, especially the terminal carboxyl group of a C-terminalamino acid, are preferably amidated with, for example, ammonia to formthe C-terminal amide, Alternatively, the terminal carboxyl group of theC-terminal amino acid may be amidated with any primary or secondaryamine. The primary or secondary amine may, for example, be an alkyl,especially a branched or unbranched C₁-C₄ alkyl, or an aryl amine.Accordingly, the amino acid at the C-terminus of the peptide may beconverted to an amido, N-methylamido, N-ethylamido, N,N-dimethylyamido,N,N-diethylamido, N-methyl-N-ethylamido N-phenylamido orN-phenyl-N-ethylamido group.

The free carboxylate groups of the asparagine glutamine, aspartic acid,and glutamic acid residues not occurring at the C-terminus of thearomatic-cationic peptides of the present invention may also be amidatedwherever they occur within the peptide. The amidation at these internalpositions may be with ammonia or any of the primary or secondary aminesdescribed above.

In one embodiment, the aromatic-cationic peptide useful in the methodsof the present invention is a tripeptide having two net positive chargesand at least one aromatic amino acid. In a particular embodiment, thearomatic-cationic peptide useful in the methods of the present inventionis a tripeptide having two net positive charges and two aromatic aminoacids.

Aromatic-cationic peptides useful in the methods of the presentinvention include, but are not limited to, the following peptideexamples:

Lys-D-Arg-Tyr-NH₂, D-Tyr-Trp-Lys-NH₂, Trp-D-Lys-Tyr-Arg-NH₂,Tyr-His-D-Gly-Met, Tyr-D-Arg-Phe-Lys-Glu-NH₂, Met-Tyr-D-Arg-Phe-Arg,D-His-Glu-Lys-Tyr-D-Phe-Arg, Lys-D-Gln-Tyr-Arg-D-Phe-Trp-NH₂,Phe-D-Arg-Lys-Trp-Tyr-D-Arg-His, Gly-D-Phe-Lys-His-D-Arg-Tyr-NH₂,Val-D-Lys-His-Tyr-D-Phe-Ser-Tyr-Arg-NH₂,Trp-Lys-Phe-D-Asp-Arg-Tyr-D-His-Lys,Lys-Trp-D-Tyr-Arg-Asn-Phe-Tyr-D-His-NH₂,Thr-Gly-Tyr-Arg-D-His-Phe-Trp-D-His-Lys,Asp-D-Trp-Lys-Tyr-D-His-Phe-Arg-D-Gly-Lys-NH₂,D-His-Lys-Tyr-D-Phe-Glu-D-Asp-D-Asp-D-His-D-Lys-Arg-Trp-NH₂,Ala-D-Phe-D-Arg-Tyr-Lys-D-Trp-His-D-Tyr-Gly-Phe,Tyr-D-His-Phe-D-Arg-Asp-Lys-D-Arg-His-Trp-D-His-Phe,Phe-Phe-D-Tyr-Arg-Glu-Asp-D-Lys-Arg-D-Arg-His-Phe-NH₂,Phe-Tyr-Lys-D-Arg-Trp-His-D-Lys-D-Lys-Glu-Arg-D-Tyr-Thr,Tyr-Asp-D-Lys-Tyr-Phe-D-Lys-D-Arg-Phe-Pro-D-Tyr-His-Lys,Glu-Arg-D-Lys-Tyr-D-Val-Phe-D-His-Trp-Arg-D-Gly-Tyr-Arg-D-Met-NH₂,Arg-D-Leu-D-Tyr-Phe-Lys-Glu-D-Lys-Arg-D-Trp-Lys-D-Phe-Tyr-D-Arg-Gly,D-Glu-Asp-Lys-D-Arg-D-His-Phe-Phe-D-Val-Tyr-Arg-Tyr-D-Tyr-Arg-His-Phe-NH₂,Asp-Arg-D-Phe-Cys-Phe-D-Arg-D-Lys-Tyr-Arg-D-Tyr-Trp-D-His-Tyr-D-Phe-Lys-Phe,His-Tyr-D-Arg-Trp-Lys-Phe-D-Asp-Ala-Arg-Cys-D-Tyr-His-Phe-D-Lys-Tyr-His-Ser-NH₂,Gly-Ala-Lys-Phe-D-Lys-Glu-Arg-Tyr-His-D-Arg-D-Arg-Asp-Tyr-Trp-D-His-Trp-His-D-Lys-Asp,andThr-Tyr-Arg-D-Lys-Trp-Tyr-Glu-Asp-D-Lys-D-Arg-His-Phe-D-Tyr-Gly-Val-Ile-D-His-Arg-Tyr-Lys-NH₂.

In one embodiment, the peptides useful in the methods of the presentinvention have mu-opioid receptor agonist activity (i.e., activate themu-opioid receptor). Activation of the mu-opioid receptor typicallyelicits an analgesic effect.

In certain instances, an aromatic-cationic peptide having mu-opioidreceptor activity is preferred. For example, during short-termtreatment, such as in an acute disease or condition, it may bebeneficial to use an aromatic-cationic peptide that activates themu-opioid receptor. For example, the acute diseases and conditions canbe associated with moderate or severe pain. In these instances, theanalgesic effect of the aromatic-cationic peptide may be beneficial inthe treatment regiment of the patient or other mammal, although anaromatic-cationic peptide which does not activate the mu-opioid receptormay also be used with or without an analgesic according to clinicalrequirements.

Alternatively, in other instances, an aromatic-cationic peptide thatdoes not have mu-opioid receptor activity is preferred. For example,during long-term treatment, such as in a chronic disease state orcondition, the use of an aromatic-cationic peptide that activates themu-opioid receptor may be contraindicated. In these instances, thepotentially adverse or addictive effects of the aromatic-cationicpeptide may preclude the use of an aromatic-cationic peptide thatactivates the mu-opioid receptor in the treatment regimen of a humanpatients or other mammal.

Potential adverse effects may include sedation, constipation, nervoussystem depression and respiratory depression. In such instancesaromatic-cationic peptide that does not activate the mu-opioid receptormay be an appropriate treatment.

Examples of acute conditions include heart attack, stroke and traumaticinjury. Traumatic injury may include traumatic brain and spinal cordinjury.

Examples of chronic diseases or conditions include coronary arterydisease and any neurodegenerative disorders, such as those describedbelow.

Peptides useful in the methods of the present invention which havemu-opioid receptor activity are typically those peptides which have atrysine residue or a tyrosine derivative at the N-terminus (i.e., thefirst amino acid position). Preferred derivatives of tyrosine include2′-methyltyrosine (Mmt); 2′,6′-dimethlyltyrosine (2′,6′Dmt),3′,5′-dimethyltryosine (3′5′Dmt); N,2′,6′-trimethyltyrosine (Tmt); and2′-hydroxy-6′-methyltryosine (Hmt).

In a particular preferred embodiment, a peptide that has mu-opioidreceptor activity has the formula Tyr-D-Arg-Phe-Lys-NH₂ (for conveniencerepresented by the acronym: DALDA, which is referred to herein as SS-01.DALDA has a net positive charge of three, contributed by the amino acidstyrosine, arginine, and lysine and has two aromatic groups contributedby the amino acids phenylalanine and tyrosine. The tyrosine of DALDA canbe a modified derivative of tyrosine such as in 2′,6′-dimethyltyrosineto produce the compound having the formula 2′,6′-Dmt-Arg-Phe-Lys-NH₂(i.e., Dmt¹-DALDA, which is referred to herein as SS-02).

Peptides that do not have mu-opioid receptor activity generally do nothave a tyrosine residue or a derivative of tyrosine at the N-terminus(i.e., amino acid position one). The amino acid at the N-terminus can beany naturally occurring or non-naturally occurring amino acid other thantyrosine.

In one embodiment, the amino acid at the N-terminus is phenylalanine orits derivative. Preferred derivatives of phenylalanine include2′-methylphenylalanine (Mmp), 2′,6′-dimethylphenylalanine (Dmp),N,2′,6′-trimethylphenylalanine (Tmp) and2′-hydroxy-6′-methylphenylalanine (Hmp). In another preferredembodiment, the amino acid residue at the N-terminus is arginine. Anexample of such a peptide is D-Arg-2′6′-Dmt-Lys-Phe-NH₂ (referred to inthis specification as SS-31).

Another aromatic-cationic peptide that does not have mu-opioid receptoractivity has the formula Phe-D-Arg-Dmt-Lys-NH₂. Alternatively, theN-terminal phenylalanine can be a derivative of phenylalanine such as2′,6′-dimethylphenylalanine (2′6′Dmp). DALDA containing2′,6′-dimethylphenylalanine at amino acid position one has the formula2′,6′-Dmp-D-Arg-Dmt-Lys-NH₂.

In a preferred embodiment, the amino acid sequence of Dmt¹-DALDA (SS-02)is rearranged such that Dmt is not at the N-terminus. An example of suchan aromatic-cationic peptide that does not have mu-opioid receptoractivity has the formula D-Arg-2′6′Dmt-Lys-Phe-NH₂ (SS-31).

DALDA, SS-31, and their derivatives can further include functionalanalogs. A peptide is considered a functional analog of DALDA or SS-31if the analog has the same function as DALDA or SS-31. The analog may,for example, be a substitution variant of DALDA or SS-31, wherein one ormore amino acid is substituted by another amino acid.

Suitable substitution variants of DALDA or SS-31 include conservativeamino acid substitutions. Amino acids may be grouped according to theirphysicochemical characteristics as follows:

(a) Non-polar amino acids: Ala(A) Ser(S) Thr(T) Pro(P) Gly(G);

(b) Acidic amino acids: Asn(N) Asp(D) Glu(E) Gln(Q);

(c) Basic amino acids: His(H) Arg(R) Lys(K);

(d) Hydrophobic amino acids: Met(M) Leu(L) Ile(I) Val(V); and

(e) Aromatic amino acids: Phe(F) Tyr(Y) Trp(W) His(H).

Substitutions of an amino acid link peptide by another amino acid in thesame group is referred to as a conservative substitution and maypreserve the physicochemical characteristics of the original peptide. Incontrast, substitutions of an amino acid in a peptide by another aminoacid in a different group is generally more likely to alter thecharacteristics of the original peptide.

Examples of analogs useful in the practice of the present invention thatactivate mu-opioid receptors include, but are not limited to, thearomatic-cationic peptides shown in Table 1.

TABLE 1 Amino Acid Amino Acid Amino Acid Amino Acid Amino Acid Position5 C-Terminal Position 1 Position 2 Position 3 Position 4 (if present)Modification Tyr D-Arg Phe Lys NH₂ Tyr D-Arg Phe Orn NH₂ Tyr D-Arg PheDab NH₂ Tyr D-Arg Phe Dap NH₂ 2′6′Dmt D-Arg Phe Lys NH₂ 2′6′Dmt D-ArgPhe Lys Cys NH₂ 2′6′Dmt D-Arg Phe Lys-NH(CH₂)₂—NH-dns NH₂ 2′6′Dmt D-ArgPhe Lys-NH(CH₂)₂—NH-atn NH₂ 2′6′Dmt D-Arg Phe dnsLys NH₂ 2′6′Dmt D-CitPhe Lys NH₂ 2′6′Dmt D-Cit Phe Ahp NH₂ 2′6′Dmt D-Arg Phe Orn NH₂ 2′6′DmtD-Arg Phe Dab NH₂ 2′6′Dmt D-Arg Phe Dap NH₂ 2′6′Dmt D-Arg PheAhp(2-aminoheptanoic acid) NH₂ Bio-2′6′Dmt D-Arg Phe Lys NH₂ 3′5′DmtD-Arg Phe Lys NH₂ 3′5′Dmt D-Arg Phe Orn NH₂ 3′5′Dmt D-Arg Phe Dab NH₂3′5′Dmt D-Arg Phe Dap NH₂ 3′5′Dmt D-Arg Tyr Lys NH₂ Tyr D-Arg Tyr OrnNH₂ Tyr D-Arg Tyr Dab NH₂ Tyr D-Arg Tyr Dap NH₂ 2′6′Dmt D-Arg Tyr LysNH₂ 2′6′Dmt D-Arg Tyr Orn NH₂ 2′6′Dmt D-Arg Tyr Dab NH₂ 2′6′Dmt D-ArgTyr Dap NH₂ 2′6′Dmt D-Arg 2′6′Dmt Lys NH₂ 2′6′Dmt D-Arg 2′6′Dmt Orn NH₂2′6′Dmt D-Arg 2′6′Dmt Dab NH₂ 2′6′Dmt D-Arg 2′6′Dmt Dap NH₂ 3′5′DmtD-Arg 3′5′Dmt Arg NH₂ 3′5′Dmt D-Arg 3′5′Dmt Lys NH₂ 3′5′Dmt D-Arg3′5′Dmt Orn NH₂ 3′5′Dmt D-Arg 3′5′Dmt Dab NH₂ Tyr D-Lys Phe Dap NH₂ TyrD-Lys Phe Arg NH₂ Tyr D-Lys Phe Lys NH₂ Tyr D-Lys Phe Orn NH₂ 2′6′DmtD-Lys Phe Dab NH₂ 2′6′Dmt D-Lys Phe Dap NH₂ 2′6′Dmt D-Lys Phe Arg NH₂2′6′Dmt D-Lys Phe Lys NH₂ 2′6′Dmt D-Lys Phe Orn NH₂ 2′6′Dmt D-Lys PheDab NH₂ 2′6′Dmt D-Lys Phe Dap NH₂ 2′6′Dmt D-Lys Phe Arg NH₂ 3′5′DmtD-Lys Phe Orn NH₂ 3′5′Dmt D-Lys Phe Dab NH₂ 3′5′Dmt D-Lys Phe Dap NH₂3′5′Dmt D-Lys Phe Arg NH₂ Tyr D-Lys Tyr Lys NH₂ Tyr D-Lys Tyr Orn NH₂Tyr D-Lys Tyr Dab NH₂ Tyr D-Lys Tyr Dap NH₂ 2′6′Dmt D-Lys Tyr Lys NH₂2′6′Dmt D-Lys Tyr Orn NH₂ 2′6′Dmt D-Lys Tyr Dab NH₂ 2′6′Dmt D-Lys TyrDap NH₂ 2′6′Dmt D-Lys 2′6′Dmt Lys 2′6′Dmt D-Lys 2′6′Dmt Orn NH₂ 2′6′DmtD-Lys 2′6′Dmt Dab NH₂ 2′6′Dmt D-Lys 2′6′Dmt Dap NH₂ 2′6′Dmt D-Arg PhednsDap NH₂ 2′6′Dmt D-Arg Phe atnDap NH₂ 3′5′Dmt D-Lys 3′5′Dmt Lys NH₂3′5′Dmt D-Lys 3′5′Dmt Orn NH₂ 3′5′Dmt D-Lys 3′5′Dmt Dab NH₂ 3′5′DmtD-Lys 3′5′Dmt Dap NH₂ Tyr D-Lys Phe Arg NH₂ Tyr D-Orn Phe Arg NH₂ TyrD-Dab Phe Arg NH₂ Tyr D-Dap Phe Arg NH₂ 2′6′Dmt D-Arg Phe Arg NH₂2′6′Dmt D-Lys Phe Arg NH₂ 2′6′Dmt D-Orn Phe Arg NH₂ 2′6′Dmt D-Dab PheArg NH₂ 3′5′Dmt D-Dap Phe Arg NH₂ 3′5′Dmt D-Arg Phe Arg NH₂ 3′5′DmtD-Lys Phe Arg NH₂ 3′5′Dmt D-Orn Phe Arg NH₂ Tyr D-Lys Tyr Arg NH₂ TyrD-Orn Tyr Arg NH₂ Tyr D-Dab Tyr Arg NH₂ Tyr D-Dap Tyr Arg NH₂ 2′6′DmtD-Arg 2′6′Dmt Arg NH₂ 2′6′Dmt D-Lys 2′6′Dmt Arg NH₂ 2′6′Dmt D-Orn2′6′Dmt Arg NH₂ 2′6′Dmt D-Dab 2′6′Dmt Arg NH₂ 3′5′Dmt D-Dap 3′5′Dmt ArgNH₂ 3′5′Dmt D-Arg 3′5′Dmt Arg NH₂ 3′5′Dmt D-Lys 3′5′Dmt Arg NH₂ 3′5′DmtD-Orn 3′5′Dmt Arg NH₂ Mmt D-Arg Phe Lys NH₂ Mmt D-Arg Phe Orn NH₂ MmtD-Arg Phe Dab NH₂ Mmt D-Arg Phe Dap NH₂ Tmt D-Arg Phe Lys NH₂ Tmt D-ArgPhe Orn NH₂ Tmt D-Arg Phe Dab NH₂ Tmt D-Arg Phe Dap NH₂ Hmt D-Arg PheLys NH₂ Hmt D-Arg Phe Orn NH₂ Hmt D-Arg Phe Dab NH₂ Hmt D-Arg Phe DapNH₂ Mmt D-Lys Phe Lys NH₂ Mmt D-Lys Phe Orn NH₂ Mmt D-Lys Phe Dab NH₂Mmt D-Lys Phe Dap NH₂ Mmt D-Lys Phe Arg NH₂ Tmt D-Lys Phe Lys NH₂ TmtD-Lys Phe Orn NH₂ Tmt D-Lys Phe Dab NH₂ Tmt D-Lys Phe Dap NH₂ Tmt D-LysPhe Arg NH₂ Hmt D-Lys Phe Lys NH₂ Hmt D-Lys Phe Orn NH₂ Hmt D-Lys PheDab NH₂ Hmt D-Lys Phe Dap NH₂ Hmt D-Lys Phe Arg NH₂ Mmt D-Lys Phe ArgNH₂ Mmt D-Orn Phe Arg NH₂ Mmt D-Dab Phe Arg NH₂ Mmt D-Dap Phe Arg NH₂Mmt D-Arg Phe Arg NH₂ Tmt D-Lys Phe Arg NH₂ Tmt D-Orn Phe Arg NH₂ TmtD-Dab Phe Arg NH₂ Tmt D-Dap Phe Arg NH₂ Tmt D-Arg Phe Arg NH₂ Hmt D-LysPhe Arg NH₂ Hmt D-Orn Phe Arg NH₂ Hmt D-Dab Phe Arg NH₂ Hmt D-Dap PheArg NH₂ Hmt D-Arg Phe Arg NH₂ Dab = diaminobutyric acid Dap =diaminopropionic acid Dmt = dimethyltyrosine Mmt = 2′-methyltyrosine Tmt= N,2′,6′-trimethyltyrosine Hmt = 2′-hydroxy,6′-methyltyrosine dnsDap =β-dansyl-L-α,β-diaminopropionic acid atnDap =β-anthraniloyl-L-α,β-diaminopropionic acid Bio = biotin

Examples of analogs useful in the practice of the present invention thatdo not activate mu-opioid receptors include, but are not limited to, thearomatic-cationic peptides shown in Table 2.

TABLE 2 Amino Acid Amino Amino Amino Amino Position 5 Acid Acid AcidAcid (if C-Terminal Position 1 Position 2 Position 3 Position 4 present)Modification D-Arg Dmt Lys Phe NH₂ D-Arg Dmt Phe Lys NH₂ D-Arg Phe LysDmt NH₂ D-Arg Phe Dmt Lys NH₂ D-Arg Lys Dmt Phe NH₂ D-Arg Lys Phe DmtNH₂ Phe Lys Dmt D-Arg NH₂ Phe Lys D-Arg Dmt NH₂ Phe D-Arg Dmt Lys NH₂Phe D-Arg Lys Dmt NH₂ Phe Dmt D-Arg Lys NH₂ Phe Dmt Lys D-Arg NH₂ LysPhe D-Arg Dmt NH₂ Lys Phe Dmt D- D-Arg NH₂ Lys Dmt Arg Phe NH₂ Lys DmtPhe D-Arg NH₂ Lys D-Arg Phe Dmt NH₂ Lys D-Arg Dmt Phe NH₂ D-Arg DmtD-Arg Phe NH₂ D-Arg Dmt D-Arg Dmt NH₂ D-Arg Dmt D-Arg Tyr NH₂ D-Arg DmtD-Arg Trp NH₂ Trp D-Arg Phe Lys NH₂ Trp D-Arg Tyr Lys NH₂ Trp D-Arg TrpLys NH₂ Trp D-Arg Dmt Lys NH₂ D-Arg Trp Lys Phe NH₂ D-Arg Trp Phe LysNH₂ D-Arg Trp Lys Dmt NH₂ D-Arg Trp Dmt Lys NH₂ D-Arg Lys Trp Phe NH₂D-Arg Lys Trp Dmt NH₂ NH₂

The amino acids of the peptides shown in table 1 and 2 may be in eitherthe L- or the D-configuration.

Methods of Reducing Oxidative Damage

The peptides described above are useful in reducing oxidative damage ina mammal in need thereof. Mammals in need of reducing oxidative damageare those mammals suffering from a disease, condition or treatmentassociated with oxidative damage. Typically, the oxidative damage iscaused by free radicals, such as reactive oxygen species (ROS) and/orreactive nitrogen species (RNS). Examples of ROS and RNS includehydroxyl radical

(HO), superoxide anion radical (O₂ ⁻) nitric oxide (NO) hydrogenperoxide (H₂O₂), hypochlorous acid (HOCl) and peroxynitrite anion.(ONOO⁻).

In one embodiment, a mammal in need thereof may be a mammal undergoing atreatment associated with oxidative damage. For example, the mammal maybe undergoing reperfusion. Reperfusion refers to the restoration ofblood flow to any organ or tissue in which the flow of blood isdecreased or blocked. The restoration of blood flow during reperfusionleads to respiratory burst and formation of free radicals.

Decreased or blocked blood flow may be due to hypoxia or ischemia. Theloss or severe reduction in blood supply during hypoxia or ischemia may,for example, be due to thromboembolic stroke, coronary atherosclerosis,or peripheral vascular disease.

Numerous organs and tissues are subject to ischemia or hypoxia. Examplesof such organs include brain, heart, kidney, intestine and prostate. Thetissue affected is typically muscle, such as cardiac, skeletal, orsmooth muscle. For instance, cardiac muscle ischemia or hypoxia iscommonly caused by atherosclerotic or thrombotic blockages which lead tothe reduction or loss of oxygen delivery to the cardiac tissues by thecardiac arterial and capillary blood supply. Such cardiac ischemia orhypoxia may cause pain and necrosis of the affected cardiac muscle, andultimately may lead to cardiac failure.

Ischemia or hypoxia in skeletal muscle or smooth muscle may arise fromsimilar causes. For example, ischemia or hypoxia in intestinal smoothmuscle or skeletal muscle of the limbs may also be caused byatherosclerotic or thrombotic blockages.

The restoration of blood flow (reperfusion) can occur by any methodknown to those in the art. For instance, reperfusion of ischemic cardiactissues may arise from angioplasty, coronary artery bypass graft, or theuse of thrombolytic drugs. Reducing oxidative damage associated withischemia/hypoxia and reperfusion is important because the tissue damageassociated with ischemia/hypoxia and reperfusion is associated with, forexample, myocardial infarction, stroke and hemorrhagic shock.

In another embodiment, a mammal in need thereof can be a mammal with adisease or condition associated with oxidative damage. The oxidativedamage can occur in any cell, tissue or organ of the mammal. Examples ofcells, tissues or organs include, but are not limited to, endothelialcells, epithelial cells, nervous system cells, skin, heart, lung, kidneyand liver. For example, lipid peroxidation and an inflammatory processare associated with oxidative damage for a disease or condition.

Lipid peroxidation refers to oxidative modification of lipids. Thelipids can be present in the membrane of a cell. This modification ofmembrane lipids typically results in change and/or damage to themembrane function of a cell. In addition, lipid peroxidation can alsooccur in lipids or lipoproteins exogenous of a cell. For example,low-density lipoproteins are susceptible to lipid per-oxidation. Anexample of a condition associated with lipid peroxidation isatherosclerosis. Reducing oxidative damage associated withatherosclerosis is important since atherosclerosis is implicated in, forexample, heart attacks and coronary artery disease.

Inflammatory process refers to the activation of the immune system.Typically, the immune system is activated by an antigenic substance. Theantigenic substance can be any substance recognized by the immunesystem, and include self-derived particles and foreign-derivedparticles. Examples of diseases or conditions occurring from aninflammatory process to self-derived particles include arthritis andmultiple sclerosis. Examples of foreign particles include viruses andbacteria.

The virus can be any virus which activates an inflammatory process, andassociated with oxidative damage. Examples of viruses include, hepatitisA, B or C virus, human immunodeficiency virus, influenza virus, andbovine diarrhea virus. For example, hepatitis virus can elicit aninflammatory process and formation of free radicals, thereby damagingthe liver.

The bacteria can be any bacteria, and include gram-negative orgram-positive bacteria. Gram-negative bacteria containlipopolysaccharide in the bacteria wall. Examples of gram-negativebacterial include Escherichia coli, Klebsiella pneumoniae, Proteusspecies, Pseudomonas aeruginosa, Serratia, and Bacteroides. Examples ofgram-positive bacteria include pneumococci and streptococci.

An example of an inflammatory process associated with oxidative stresscaused by a bacteria is sepsis. Typically, sepsis occurs whengram-negative bacteria enter the bloodstream.

Liver damage caused by a toxic agent is another condition associatedwith an inflammatory process and oxidative stress. The toxic agent canbe any agent which causes damage to the liver. For example, the toxicagent can cause apoptosis and/or necrosis of liver cells. Examples ofsuch agents include alcohol, and medication, such as prescription andnon-prescription drugs taken to treat a disease or condition.

The methods of the present invention can also be used in reducingoxidative damage associated with any neurodegenerative disease orcondition. The neurodegenerative disease can affect any cell, tissue ororgan of the central and peripheral nervous system. Examples of suchcells, tissues and organs include, the brain, spinal cord, neurons,ganglia, Schwann cells, astrocytes, oligodendrocytes and microglia.

The neurodegenerative condition can be an acute condition, such as astroke or a traumatic brain or spinal cord injury. In anotherembodiment, the neurodegenerative disease or condition can be a chronicneurodegenerative condition. In a chronic neurodegenerative condition,the free radicals can, for example, cause damage to a protein. Anexample of such a protein is amyloid β-protein. Examples of chronicneurodegenerative diseases associated with damage by free radicalsinclude Parkinson's disease, Alzheimer's disease, Huntington's diseaseand Amyotrophic Lateral Sclerosis (also known as Lou Gherig's disease).

Other conditions which can be treated in accordance with the presentinvention include preeclampsia, diabetes, and symptoms of and conditionsassociated with aging, such as macular degeneration, wrinkles.

In another embodiment, the peptides useful in the present invention mayalso be used in reducing oxidative damage in an organ of a mammal priorto transplantation. For example, a removed organ, when subjected toreperfusion after transplantation can be susceptible to oxidativedamage. Therefore, the peptides can be used to reduce oxidative damagefrom reperfusion of the transplanted organ.

The removed organ can be any organ suitable for transplantation.Examples of such organs include, the heart, liver, kidney, lung, andpancreatic islets. The removed organ is placed in a suitable medium,such as in a standard buffered solution commonly used in the art.

For example, a removed heart can be placed in a cardioplegic solutioncontaining the peptides described above. The concentration of peptidesin the standard buffered solution can be easily determined by thoseskilled in the art. Such concentrations may be, for example, betweenabout 0.01 nM to about 10 μM, preferably about 0.1 nM to about 10 μM,more preferably about 1 μM to about 5 μM, and even more preferably about1 nM to about 100 nM.

In yet another embodiment, the invention provides a method for reducingoxidative damage in a cell in need thereof. Cells in need of reducingoxidative damage are generally those cells in which the cell membrane orDNA of the cell has been damaged by free radicals, for example, ROSand/or RNS. Examples of cells capable of being subjected to oxidativedamage include the cells described herein. Suitable examples of cellsinclude pancreatic islet cells, myocytes, endothelial cells, neuronalcells, stem cells, etc.

The cells can be tissue culture cells. Alternatively the cells may beobtained from a mammal. In one instance, the cells can be damaged byoxidative damage as a result of an insult. Such insults include, forexample, a disease or condition (e.g., diabetes, etc.) or ultravioletradiation (e.g., sun, etc.). For example pancreatic islet cells damagedby oxidative damage as a result of diabetes can be obtained from amammal.

The peptides described above can be administered to the cells by anymethod known to those skilled in the art. For example, the peptides canbe incubated with the cells under suitable conditions. Stick conditionscan be readily determined by those skilled in the art.

Due to reduction of oxidative damage, the treated cells may be capableof regenerating. Such regenerated cells may be administered back intothe mammal as a therapeutic treatment for a disease or condition. Asmentioned above, one such condition is diabetes.

Oxidative damage is considered to be “reduced” if the amount ofoxidative damage in a mammal, a removed organ, or a cell is decreasedafter administration of an effective amount of the aromatic cationicpeptides described above. Typically, the oxidative damage is consideredto be reduced if the oxidative damage is decreased by at least about10%, preferably at least about 25%, more preferably at least about 50%,even more preferably at least about 75%, and most preferably at leastabout 90%.

Synthesis of the Peptides

The peptides useful in the methods of the present invention may bechemically synthesized by any of the methods well known in the art.Suitable methods for synthesizing the protein include, for example thosedescribed by Stuart and Young in “Solid Phase Peptide Synthesis,” SecondEdition, Pierce Chemical Company (1984), and in “Solid Phase PeptideSynthesis,” Methods Enzymol. 289, Academic Press, Inc, New York (1997).

Modes of Administration

The peptide useful in the methods of the present invention isadministered to a mammal in an amount effective in reducing oxidativedamage. The effective amount is determined during pre-clinical trialsand clinical trials by methods familiar to physicians and clinicians.

An effective amount of a peptide useful in the methods of the presentinvention, preferably in a pharmaceutical composition, may beadministered to a mammal in need thereof by any of a number ofwell-known methods for administering pharmaceutical compounds.

The peptide may be administered systemically or locally. In oneembodiment, the peptide is administered intravenously. For example, thearomatic-cationic peptides useful in the methods of the presentinvention may be administered via rapid intravenous bolus injection.Preferably, however, the peptide is administered as a constant rateintravenous infusion.

The peptide can be injected directly into coronary artery during forexample, angioplasty of or coronary bypass surgery, or applied ontocoronary stents.

The peptide may also be administered orally, topically, intranasally,intramuscularly, subcutaneously, or transdermally. In a preferredembodiment, transdermal administration of the aromatic-cationic peptidesby methods of the present invention is by iontophoresis, in which thecharged peptide is delivered across the skin by an electric current.

Other routes of administration include intracerebroventricularly orintrathecally. intracerebroventiculatly refers to administration intothe ventricular system of the brain. Intrathecally refers toadministration into the space under the arachnoid membrane of the spinalcord. This intracerebroventicular or intrathecal administration may bepreferred for those diseases and conditions which affect the organs ortissues of the central nervous system. In a preferred embodiment,intrathecal administration is used for traumatic spinal cord injury.

The peptides useful in the methods of the invention may also beadministered to mammals by sustained release as is known in the art.Sustained release administration is a method of drug delivery to achievea certain level of the drug over a particular period of time. The leveltypically is measured by serum or plasma concentration.

A description of methods for delivering a compound by controlled releasecan be found in PCT Application No. WO 02/083106. The PCT application isincorporated herein by reference in its entirety.

Any formulation known in the art of pharmacy is suitable foradministration of the aromatic-cationic peptides useful in the methodsof the present invention. For oral administration, liquid or solidformulations may be used. Some examples of formulations include tablets,gelatin capsules, pills, troches, elixirs, suspensions, syrups, wafers,chewing gum and the like. The peptides can be mixed with a suitablepharmaceutical carrier (vehicle) or excipient as understood bypractitioners in the art. Examples of carriers and excipients includestarch, milk, sugar, certain types of clay, gelatin, lactic acid,stearic acid or salts thereof, including magnesium or calcium stearate,talc, vegetable fats or oils, gums and glycols.

For systemic, intracerebroventricular, intrathecal, topical, intranasal,subcutaneous, or transdermal administration, formulations of thearomatic-cationic peptides useful in the methods of the present inventoils may utilize conventional diluents, carriers, or excipients etc.,such as are known in the art can be employed to deliver the peptides.For example, the formulations may comprise one or more of the following:a stabilizing a surfactant, preferably a nonionic surfactant, andoptionally a salt and/or a buffering agent. The peptide may be deliveredin the form of an aqueous solution, or in a lyophilized form.

The stabilizer may, for example, be an amino acid, such as for instance,glycine; or an oligosaccharide, such as for example, sucrose, tetralose,lactose or a dextran. Alternatively, the stabilizer may be a sugaralcohol, such as for instance, mannitol; or a combination thereof.Preferably the stabilizer or combination of stabilizers constitutes fromabout 0.1% to about 10% weight for weight of the peptide.

The surfactant is preferably a nonionic surfactant, such as apolysorbate. Some examples of suitable surfactants include Tween20,Tween80; a polyethylene glycol or a polyoxyethylene polyoxypropyleneglycol, such as Pluronic F-68 at from about 0.001% (w/v) to about 10%(w/v).

The salt or buttering agent may be any salt or buffering agent such asfor example, sodium chloride, or sodium/potassium phosphate,respectively. Preferably, the buttering agent maintains the pH of thepharmaceutical composition in the range of about 5.5 to about 7.5. Thesalt and/or buffering agent is also useful to maintain the osmolality ata level suitable for administration to a human or an animal. Preferablythe salt or buffering agent is present at a roughly isotonicconcentration of about 150 mM to about 300 mM.

The formulations of the peptides useful in the methods of the presentinvention may additionally contain one or more conventional additive.Some examples of such additives include a solubilizer such as, forexample, glycerol; an antioxidant such as for example, benzalkoniumchloride (a mixture of quaternary ammonium compounds, known as “quats”),benzyl alcohol, chloretone or chlorobutanol; anaesthetic agent such asfor example a morphine derivative; or an isotonic agent etc., such asdescribed above. As a further precaution against oxidation or otherspoilage, the pharmaceutical compositions may be stored under nitrogengas in vials sealed with impermeable stoppers.

The mammal treated in accordance with the invention can be any mammal,including, for example, farm animals, such as sheep, pigs, cows, andhorses pet animals such as dogs and cats, laboratory animals, such asrats, mice and rabbits. In a preferred embodiment, the mammal is ahuman.

EXAMPLES Example 1 [Dmt¹]DALDA Penetrates Cell Membrane

The cellular uptake of [³H][Dmt¹]DALDA was studied using a humanintestinal epithelial cell line (Caco-2), and confirmed with SH-SY5Y(human neuroblastoma cell), HEK293 (human embryonic kidney cell) andCRFK cells (kidney epithelial cell). Monolayers of cells were grown on12-well plates (5×10⁵ cells/well) coated with collagen for 3 days. Onday 4, cells were washed twice with pre-warmed HBSS, and then incubatedwith 0.2 ml of HBSS containing either 250 nM [³H][Dmt¹]DALDA at 37° C.for 4° C. for various times up to 1 h.

[³H][Dmt¹]DALDA was observed in cell lysate as early as 5 min, andsteady state levels were achieved by 30 min. The total amount of[³H][Dmt¹]DALDA recovered in the cell lysate after 1 h incubationrepresented about 1% of the total drug. The uptake of [³H][Dmt¹]DALDAwas slower at 4° C. compared to 37° C., but reached 76.5% by 45 min and86.3% by 1 h. The internalization of [³H][Dmt¹]DALDA was not limited toCaco-2 cells, but was also observed in SH-SY5Y, HEK293 and CRFK cells.The intracellular concentration of [Dmt¹]DALDA was estimated to beapproximately 50 times higher than extracellular concentration.

In a separate experiment, cells were incubated with a range of[Dmt¹]DALDA concentrations (1 μM-3 mM) for 1 h at 37° C. At the end ofthe incubation period, cells were washed 4 times with HBSS, and 0.2 mlof 0.1N NaOH with 1% SDS was added to each well. The cell contents werethen transferred to scintillation vials and radioactivity counted. Todistinguish between internalized radioactivity from surface-associatedradioactivity, an acid-wash step was included. Prior to cell lysis,cells were incubated with 0.2 ml of 0.2 M acetic acid/0.05 M NaCl for 5min on ice.

The uptake of [Dmt¹]DALDA into Caco-2 cells was confirmed by confocallaser scanning microscopy (CLSM) using a fluorescent analog of[Dmt¹]DALDA (Dmt-D-Arg-Phe-dnsDap-NH₂; wherednsDap=β-dansyl-1-α,β-diaminopropionic acid). Cells were grown asdescribed above and were plated on (35 mm) glass bottom dishes (MatTekCorp., Ashland, Mass.) for 2 days. The medium was then removed and cellswere incubated with 1 ml of HBSS containing 0.1 μM to 1.0 μM of thefluorescent peptide analog at 37° C. for 1 h. Cells were then washedthree times with ice-cold HBSS and covered with 200 μl of PBS, andmicroscopy was performed within 10 min at room temperature using a Nikonconfocal laser scanning microscope with a C-Apochromat 63×/1.2W corrobjective. Excitation was performed at 340 nm by means of a UV laser,and emission was measured at 520 nm. For optical sectioning inz-direction, 5-10 frames with 2.0 μm were made.

CLSM confirmed the uptake of fluorescent Dmt-D-Arg-Phe-dnsDap-NH₂ intoCaco-2 cells after incubation with 0.1 μM [Dmt¹,DnsDap⁴]DALDA for 1 h at37° C. The uptake of the fluorescent peptide was similar at 37° C. and4° C. The fluorescence appeared diffuse throughout the cytoplasm but wascompletely excluded from the nucleus.

Example 2—Targeting of [Dmt¹]DALDA to Mitochondria

To examine the subcellular distribution of [Dmt¹]DALDA, the fluorescentanalog, [Dmt¹, AtnDap⁴]DALDA (Dmt-D-Arg-Phe-atnDap-NH₂; whereatn=β-anthraniloyl-1-α,β-diamino-propionic acid) was prepared. Theanalog contained β-anthraniloyl-1-α,β-diaminopropionic acid in place ofthe lysine reside at position 4. The cells were grown as described inExample 1 and were plated on (35 mm) glass bottom dishes (MatTek Corp.,Ashland, Mass.) for 2 days. The medium was then removed and cells wereincubated with 1 ml of HBSS containing 0.1 μM of [Dmt¹,AtnDap⁴]DALDA at37° C. for 15 min to 1 h.

Cells were also incubated with tetramethylrhodamine methyl ester (TMRM,25 nM), a dye for staining mitochondria, for 15 min at 37° C. Cells werethen washed three times with ice-cold HBSS and covered with 200 μl ofPBS, and microscopy was performed within 10 min at room temperatureusing a Nikon confocal laser scanning microscope with a C-Apochromat63×/1.2W corr objective.

For [Dmt¹,AtnDap⁴]DALDA, excitation was performed at 350 nm by means ofa UV laser, and emission was measured at 520 nm. For TMRM, excitationwas performed at 356 nm, and emission was measured at 560 nm.

CLSM showed the uptake of fluorescent [Dmt¹,AtnDap⁴]DALDA into Caco-2cells after incubation for as little as 15 min at 37° C. The uptake ofdye was completely excluded from the nucleus, but the blue dye showed astreaky distribution within the cytoplasm. Mitochondria were labeled redwith TMRM. The distribution of [Dmt¹,AtnDap⁴]DALDA to mitochondria wasdemonstrated by the overlap of the [Dmt¹,AtnDap⁴]DALDA distribution andthe TMRM distribution.

Example 3—Scavenging of Hydrogen Peroxide by SS-02 and SS-05 (FIG. 1)

Effect of SS-02 and SS-05 (Dmt-D-Arg-Phe Orn-NH₂) on H₂O₂ as measured byluminol-induced chemiluminescence, 25 μM luminol and 0.7 IU horseradishperoxidase were added to the solution of H₂O₂ (4.4 nmol) and peptide,and chemiluminescence was monitored with a Chronolog Model 560aggregometer (Havertown, Pa.) for 20 min at 37° C.

Results show that SS-02 and SS-05 dose-dependently inhibited the luminolresponse suggesting that these peptides can scavenge H₂O₂.

Example 4—Inhibition of Lipid Peroxidation (FIG. 2)

Linoleic acid peroxidation was induced by a water-soluble initiator.ABAP (2,2′-azobis(2-amidinopropane)), and lipid peroxidation wasdetected by the formation of conjugated dienes, monitoredspectrophotometrically at 236 nm (B. Longoni, W. A. Pryor, P.Marchiafava, Biochem. Biophys. Res. Commun. 233, 778-780 (1997)).

5 ml of 0.5 M ABAP and varying concentrations of SS-02 were incubated in2.4 ml linoleic acid suspension until autoxidation rate became constant.Results show that SS-02 dose-dependently inhibited the peroxidation oflinoleic acid.

Various peptides were added in concentration of 100 μM. The data arepresented as the slope of diene formation. With the exception of SS-20(Phe-D-Arg-Phe-Lys-NH₂), SS-21 (Cyclohexyl-D-Arg-Phe-Lys-NH₂) and SS-22(Ala-D-Arg-Phe-Lys-NH₂), all other SS peptides reduced the rate oflinoleic acid peroxidation. Note that SS-20, SS-21 and SS-22 do notcontain either tyrosine or dimethyltyrosine residues. SS-01, whichcontains Tyr rather than Dmt is not as effective in preventing linoleicacid peroxidation. SS-29 is Dmt-D-Cit-Phe Lys-NH₂. SS-30 isPhe-D-Arg-Dmt-Lys-NH₂, SS-32 is Dmt-D-Arg-Phe-Ahp(2-aminoheptanoicacid)-NH₂.

Example 5—Inhibition of LDL Oxidation (FIG. 3)

Human LDL (low density lipoprotein) was prepared fresh from storedplasma. LDL oxidation was induced catalytically by the addition of 10 mMCuSO₄, and the formation of conjugated dienes was monitored at 24 nm for5 h at 37° C. (B. Moosmann and C. Behl, Mol Pharmacol., 61, 260-268(2002)).

(A) Results show that SS-02 dose-dependently inhibited the rate of LDLoxidation.

(B) Various peptides were added in concentration of 100 μM. With theexception of SS-20 (Phe-D-Arg-Phe-Lys NH₂)SS-21.(Cyclohexyl-D-Arg-Phe-Lys-NH₂) and SS-22 (Ala-D-Arg-Phe-Lys-NH₂), allother SS peptides reduced the rate of linoleic acid peroxidation(reduced rate of formation of conjugated dienes). Note that SS-20, SS-21and SS-22 do not contain either tyrosine or dimethyltyrosine residues.SS-29 is Dmt-D-Cit-Phe-Lys-NH₂, SS-30 is Phe-D-Arg-Dmt-Lys-NH₂, SS-32 isDmt-D-Arg-Phe-Ahp(2-aminoheptanoic acid)-NH₂.

Example 6—Hydrogen Peroxide Production by Isolated Mouse LiverMitochondria (FIG. 4)

Because mitochondria are a major source of ROS production, the effect ofSS-02 on H₂O₂ formation in isolated mitochondria under basal conditionsas well as after treatment with antimycin, a complex III inhibitor wasexamined. Livers were harvested from mice and homogenized in ice-coldbuffer and centrifuged at 13800×g for 10 m. The pellet was washed onceand then re-suspended in 0.3 ml of wash butter and placed on ice untiluse H₂O₂ was measured using luminol chemiluminescence as describedpreviously (Y. Li, H. Zhu, M. A. Trush, Biochim. Biophys. Acta 1428,1-12 (1999)). 0.1 mg mitochondrial protein was added to 0.5 ml potassiumphosphate buffer (100 mM, pH 8.0) in the absence or presence of SSpeptides (100 μM). 25 mM luminol and 0.7 IU horseradish peroxidase wereadded, and chemilumunescence was monitored with a Chronolog Model 560aggregometer (Havertown, Pa.) for 20 min at 37° C. The amount of H₂O₂produced was quantified as the area under the curve (AUC) over 20 min,and all data were normalized to AUC produced by mitochondria alone.

(A) The amount of H₂O₂ production was significantly reduced in thepresence of 10 μM SS-02. Addition of antimycin (1 μM) significantlyincreased H₂O₂ production by isolated mitochondria, and the increase wascompletely blocked by 1.0 μM Dmt¹-DALDA (also referred to as dDALDA inthe specification).

(B) The amount of H₂O₂ generated was significantly reduced by peptidesSS-02, SS-29, SS-30 and SS-31. SS-21 and SS-22 had no effect on H₂O₂production. Note that SS-21 and SS-22 do not contain a tyrosine ordimethyltryosine residue. The amino acid Dmt dimethyltyrosine) alonealso inhibited H₂O₂ generated.

Example 7—SS-31 Inhibits H₂O₂ Generation by Isolated Mitochondria (FIG.5)

H₂O₂ was measured using luminol chemiluminescence as describedpreviously (Y, Li, H. Zhu, M. A. Trush, Biochim. Biophys. Acta 1428,1-12 (1999)). 0.1 mg mitochondrial protein was added to 0.5 ml potassiumphosphate buffer (100 mM, pH 8.0) in the absence or presence of SS-31.25 mM luminol and 0.7 IU horseradish peroxidase were added, andchemilumunescence was monitored with a Chronolog Model 560 aggregometer(Havertown, Pa.) for 20 min at 37° C. The amount of H₂O₂ produced wasquantified as the area under the curve (AUC) over 20 min, and all datawere normalized to AUC produced by mitochondria alone.

(A)SS-31 dose-dependently reduced the spontaneous production of H₂O₂ byisolated mitochondria.

(B)SS-31 dose-dependently reduced the production of H₂O₂ induced byantimycin in isolated mitochondria.

Example 8—SS-02 and SS-31 Reduced Intracellular ROS and Increased CellSurvival (FIG. 6)

To show that the claimed peptides are effective when applied to wholecells, neuronal N₂A cells were plated in 96-well plates at a density of1×10⁴/well and allowed to grow for 2 days before treatment with tBHP(0.5 or 1 mM) for 40 min. Cells were washed twice and replaced withmedium alone or medium containing varying concentrations of SS-02 orSS-31 for 4 hr. Intracellular ROS was measured by carboxy-H2DCFDA(Molecular Probes, Portland, Oreg.). Cell death was assessed by a cellproliferation assay (MTS assay, Promega, Madison, Wis.).

Incubation with tBHP resulted in dose-dependent increase inintracellular ROS (A) and decrease in cell viability, (B and C).Incubation of these cells with either SS-31 or SS-02 dose-dependentlyreduced intracellular ROS (A) and increased cell survival (B and C),with EC50 in the nM range.

Example 9—SS-31 Prevented Loss of Cell Viability (FIG. 7)

Neuronal N₂A and SH-SY5Y cells were plated in 96-well plate at a densityof 1×10⁴/well and allowed to grow for 2 days before treatment witht-butyl hydroperoxide (tBHP) (0.05-0.1 mM) with or without SS-31 (10⁻12M to 10⁻9 M for 24 h. Cell death was assessed by a cell proliferationassay (MTS assay, Promega, Madison, Wis.).

Treatment of N₂A and SH-SY5Y cells with low doses of t-BHP (0.05-0.1 mM)for 24 h resulted in a decrease in cell viability. (A) 0.05 mM t-BHPinduced 50% loss of cell viability in N₂A cells and 30% in SH-SY5Ycells. (B) 0.1 mM t-BHP resulted in a greater reduction in cellviability in SH-SY5Y cells. Concurrent treatment of cells with SS-31resulted in a dose-dependent reduction of t-BHP-induced cytotoxicity.Complete protection against t-BHP was achieved by 1 nM SS-31.

Example 10—SS-31 Decreased Capase Activity (FIG. 8)

N₂A cells were grown on 96-well plates, treated with t-BHP (0.05 mM) inthe absence or presence of SS-31 (10⁻¹¹ M−10⁻⁸ M) at 37° C. for 12-24 h.All treatments were carried out in quadriplicates. N₂A cells wereincubated with t-BHP (50 mM) with or without SS-31 at 37° C. for 12 h.Cells were gently lifted from the plates with a cell detachment solution(Accutase, Innovative Cell Technologies, Inc., San Diego, Calif.) andwashed twice in PBS. Caspase activity was assayed using the FLICA kit(Immunochemistry Technologies LLC, Bloomington, Minn.) According to themanufacturer's recommendation, cells were resuspended (approx. 5×10⁶cells/ml) in PBS and labeled with pan-caspase inhibitor FAM-VAD-FMK for1 h at 37° C. under 5% CO₂ and protected from the light. Cells were thenrinsed to remove the unbound reagent and fixed. Fluorescence intensityin the cells was measured by a laser scanning cytometer (Beckman-CoulterXL, Beckman Coulter, Inc., Fullerton, Calif.) using the standardemission filters for green (FL1). For each run, 10,000 individual eventswere collected and stored in list-mode files for off-line analysis.

Caspase activation is the initiating trigger of the apoptotic cascade,and our results showed a significant increase in caspase activity afterincubation of SH-SY5Y cells with 50 mM t-BHP for 12 h which wasdose-dependently inhibited by increasing concentrations of SS-31.

Example 11—SS-31 Reduced Rate of ROS Accumulation (FIG. 9)

Intracellular ROS was evaluated using the fluorescent probe DCFH-DA(5-(and -6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate). DCFH-DAenters cells passively and is then deacetylated to nonfluorescent DCFH.DCFH reacts with ROS to form DCF, the fluorescent product. N₂A cells in96 sell plates were washed with HBSS and loaded with 10 μM of DCFDA for30 min. for 30 min. at 37° C. Cells were washed 3 times with BSS andexposed to 0.1 mM of t-BHP, alone or with SS-31. The oxidation of DCFwas monitored in real time by a fluorescence microplate reader(Molecular Devices) using 485 nm for excitation and 530 nm for emission.

The rate of ROS accumulation in N₂A cells treated with 0.1 mM t-BHP wasdose-dependently inhibited by the addition of SS-31.

Example 12—SS-31 Inhibited Lipid Peroxidation in Cells Exposed toOxidative Damage (FIG. 10)

SS-31 inhibited lipid peroxidation in N₂A cells treated with t-BHP.Lipid peroxidation was evaluated by measuring HNE Michael adducts. 4-HNEis one of the major aldehydic products of the peroxidation of membranepolyunsaturated fatty acids. N₂A cells were seeded on glass bottom dish1 day before t-BHP treatment (1 mM, 3 h, 37° C., 5% CO₂) in the presenceof absence of SS-31 (10⁻⁸ to 10⁻¹⁰ M). Cells were then washed twice withPBS and fixed 30 min with 4% paraformaldehyde in PBS at RT and thenwashed 3 times with PBS. Cells were then permeabilized, treated withrabbit-anti-HNE antibody followed by the secondary antibody (goatanti-rabbit IgG conjugated to biotin). Cells were mounted in Vectashieldand imaged using a Zeiss fluorescence microscope using an excitationwavelength of 460±20 nm and a longpass filter of 505 nm for emission.

(A) Untreated cells (B) cells treated with 1 mM t-BHP for 3 h; (C) cellstreated with 1 mM t-BHP and 10 nM SS-31 for 3 h.

Example 13—SS-02 Inhibits Loss of Mitochondrial Potential in CellsExposed to Hydrogen Peroxide

Caco-2 cells were treated with tBHP (1 mM) in the absence or presence ofSS-02 (0.1 μM) for 4 h, and then incubated with TMRM and examined underLSCM. In control cells, the mitochondria are clearly visualized as finestreaks throughout the cytoplasm. In cells treated with tBHP, the TMRMfluorescence is much reduced, suggesting generalized depolarization. Incontrast, concurrent treatment with SS-02 protected againstmitochondrial depolarization caused by tBHP.

Example 14—SS-31 Prevents Loss of Mitochondrial Potential and IncreasedROS Accumulation in N2A Cells Caused by Exposure to t-BHP (FIG. 11)

N₂A cells in glass bottom dish were treated with 0.1 mM t-BHP, alone orwith 1 nM SS-31, for 6 h. Cells were then loaded with 10 μm ofdichlorofluorescin (ex/em=−485/530) for 30 min at 37° C., 5% CO₂. Thencells were subjected 3 times wash with HBSS and stained with 20 nM ofMitotracker TMRM (ex/em=550/575 nm) for 15 min at 37° C. and examined byconfocal laser scanning microscopy.

Treatment of N₂A cells with t-BHP resulted in loss of TMRM fluorescenceindicating mitochondrial depolarization. There was also a concomitantincrease in DCF fluorescence indicating increase in intracellular ROS.Concurrent treatment with 1 nM SS-31 prevented mitochondrialdepolarization and reduced ROS accumulation.

Example 15—SS-31 Prevents Apoptosis Caused by Oxidative Stress (FIG. 12)

SH-SY5Y cells were grown on 96-well plates, treated with t-BHP (0.025mM) in the absence or presence of SS-31 (10⁻¹² M−10⁻⁹ M) at 37° C. for24 h. All treatments were carried out in quadriplicates. Cells were thenstained with 2 mg/ml Hoechst 33342 for 20 min, fixed with 4%paraformaldehyde, and imaged using a Zeiss fluorescent microscope(Axiovert 200M) equipped with the Zeiss Acroplan ×20 objective. Nuclearmorphology was evaluated using an excitation wavelength of 350±10 nm anda longpass filter of 400 nm for emission. All images were processed andanalyzed using the MetaMorph software (Universal Imaging Corp., WestChester, Pa.). Uniformly stained nuclei were scored as healthy, viableneurons, while condensed or fragmented nuclei were scored as apoptotic.

SS-31 prevents apoptosis induced by a low dose of t-BHP. Apoptosis wasevaluated by confocal microscopy with the fluorescent probe Hoechst33342 (A1) a representative field of cells not treated with t-BHP. (A2)Fluorescent image showing a few cells with dense, fragmented chromatinindicative of apoptotic nuclei. (A3) A representative field of cellstreated with 0.025 mM t-BHP for 24 h. (A4) Fluorescent image showing anincreased number of cells with apoptotic nuclei. (A5) A representativefield of cells treated with 0.025 mM t-BHP and 1 nM SS-31 for 24 h. (A6)Fluorescent image showing a reduced number of cells with apoptoticnuclei.

(B)SS-31 dose-dependently reduced the percent of apoptotic cells causedby 24 h treatment with a low dose of t-BHP (0.05 mM).

Example 16—SS-31 Prevents Lipid Peroxidation in Hearts Subjected toBrief Intervals of Ischemia-Reperfusion. (FIG. 13)

Isolated guinea pig hearts were perfused in a retrograde manner in aLangendorff apparatus and subjected to various intervals ofischemia-reperfusion. Hearts were then fixed immediately and embedded inparaffin. Immunohistochemical analysis of 4-hydroxy-2-nonenol(HNE)-modified proteins in the paraffin sections was carried out usingan anti-HNE antibody.

(A) Immunohistochemical analysis of 4-hydroxy-2-nonenol (HNE)-modifiedproteins in paraffin sections from guinea pig hearts aerobicallyperfused 30 min with (a) buffer; (b) 100 nM SS-02; (c) 100 nM SS-20 and(d) 1 nM SS-31, then subjected to 30 min ischemia and reperfused for 90min with same peptides. Tissue slices were incubated with anti-HNEantibody. (e) Background control: staining without primary antibody.

(B) Immunohistochemical analysis of HNE-modified proteins in paraffinsections from guinea pig hearts aerobically perfused 30 min with buffer;then subjected to 30 min ischemia and reperfused with (a) buffer; (b)100 nM SS-02; (c) 100 nM SS-20 and (d) 1 nM SS-31 for 90 min with samepeptides. Tissue slices were incubated with anti-HNE antibody. (e)Background control staining without primary antibody.

Example 17—SS-31 Increases Coronary Flow and Reduces Lipid Peroxidationand Apoptosis in Hearts Subjected to Prolonged Cold Ischemia Followed byWarm Reperfusion (FIG. 14)

Isolated guinea pig hearts were perfused in a retrograde manner in aLangendorff apparatus with a cardioplegic solution (St. Thomas solution)without or with SS-31 (1 nM) for 3 min. and then clamped and stored at4° C. for 18 h. Subsequently, the hearts were remounted in theLangendorff apparatus and reperfused with Krebs-Henseleit solution at34° C. for 90 min. Hearts were then rapidly fixed and paraffin-embedded.

(A)SS-31 significantly improved coronary flow in hearts after 18 h coldischemic storage. The shaded area represents 18 h of cold ischemia.

(B) Immunohistochemical analysis of HNE-modified proteins in paraffinsections from guinea pig hearts stored without (a) or with (b)SS-31 (1nM). (c) Background staining without primary antibody.

(C)SS-31 prevents apoptosis in endothelial cells and myocytes inisolated guinea pig hearts subjected to warm reperfusion after prolonged(18 h) cold ischemia. Apoptosis was assessed by the TUNEL stain (green)and nuclei are visualized by DAPI (blue).

Example 18—SS-31 Improves Survival of Islet Cells Isolated from MousePancreas (FIG. 15)

(A)SS-31 improves mitochondrial potential in islet cells isolated frommouse pancreas. Pancreas was harvested from mice and islet cells wereprepared according standard procedures. In some studies, SS-31 (1 nM)was added to all isolation buffers used throughout the isolationprocedure. Mitochondrial potential was measured using TMRM (red) andvisualized by confocal microscopy.

(B)SS-31 reduces apoptosis and increases viability in islet cellsisolated from mouse pancreas. Pancreas was harvested from mice and isletcells were prepared according standard procedures. In some studies,SS-31 (1 nM) was added to all isolation buffers used throughout theisolation procedure. Apoptosis was ascertained by flow cytometry usingannexin V and necrosis by propidium iodide.

Example 19—SS-31 Protects Against Oxidative Damage in Pancreatic IsletCells (FIG. 16)

Mouse pancreatic islet cells were untreated (a), or treated with 25 μMtBHP without or with 1 nM SS-31 (c). Mitochondrial potential wasmeasured by TMRM (red) and reactive oxygen species were measured by DCF(green) using confocal microscopy.

Example 20—SS-31 Protects Against Parkinson's Disease (FIG. 17)

MPTP is a neurotoxin that selectively destroys striatal dopamine neuronsand can be used as an animal model of Parkinson's Disease. MPP⁺, ametabolite of MPTP, targets mitochondria, inhibits complex I of theelectron transport chain and increases ROS production. MPP⁺ is used incell culture studied because cells are unable to metabolize MPTP to theactive metabolite. MPTP is used for animal studies.

(A)SS-31 protects dopamine cells against MPP⁺ toxicity. SN-4741 cellswere treated with buffer, 50 μM MPP⁺ or 50 μM MPP⁺ and 1 nM SS-31, for48 h, and the incidence of apoptosis was determined by fluorescentmicroscopy with Hoechst 33342. The number of condensed fragmented nucleiwas significantly increased by MPP+ treatment. Concurrent treatment withSS-31 reduced the number of apoptotic cells.

(B)SS-31 dose-dependently prevented loss of dopamine neurons in micetreated with MPTP. Three doses of MPTP (10 mg/kg) was given to mice(n=12) 2 h apart. SS-31 was administered 30 min before each MPTPinjection, and at 1 h and 12 h after the last MPTP injection. Animalswere sacrificed one week later and striatal brain regions wereimmunostained for tyrosine hydroxylase activity.

(C)SS-31 dose-dependently increased striatal dopamine DOPAC(3,4-dihydroxyphenylacetic acid) and HVA (homovanillic acid) levels inmice treated with MPTP. Three doses of MPTP (10 mg/kg) was given to mice(n=12) 2 h apart. SS-31 was administered 30 min before each MPTPinjection, and at 1 h and 12 h after the last MPTP injection.

Animals were sacrificed one week later and dopamine, DOPAC and HVAlevels were quantified by high pressure liquid chromatography.

Example 21—SS-31 Protected SH-SY5Y and N2A Cells Against tBHP InducedCytotoxicity (FIG. 18)

The loss of cell viability induced by 100 μM tBHP was accompanied by asignificant increase in LDH release in SH-SY5Y (FIG. 18A) and N₂A cell(FIG. 18B). (Concurrent treatment of cells with SS-31 resulted indose-dependent decrease in LDH release in both SH-SY5Y (P<0.01) and N₂Acells (P<0.0001). LDH release was reduced significantly by 0.1 and 1 nMof SS-31 in both cell lines (P<0.05). SS-20, the control non-scavengingpeptide, did not protect against tBHP-induced cytotoxicity in N2A cells(FIG. 18B).

Example 22—SS-31 Protected Against tBHP-Induced Apoptosis. (FIGS. 19 and20)

The translocation of phosphatidylserine from the inner leaflet of theplasma membrane to the outer leaflet is observed early in the initiationof apoptosis. This can be observed with Annexin V, a phospholipidbinding protein with high affinity for phosphatidylserine. FIG. 19Ashows that untreated N2A cells showed little to no Annexin V staining(green). Incubation of N2A cells with 50 mM tBHP for 6 h resulted inAnnexin V staining on the membranes of most cells (FIG. 19B). Combinedstaining with Annexin V and propidium iodide (red) showed many lateapoptotic cells (FIG. 19B). Concurrent treatment of N2A cells with 1 nMSS-31 and 50 μM tBHP resulted in a reduction in Annexin V− positivecells and no propidium iodide staining (FIG. 19C), suggesting that SS-31protected against tBHP-induced apoptosis.

The morphological appearance of cells treated with tBHP was alsoconsistent with apoptosis. N2A cells incubated with 50 μM tBHP for 12 hbecame rounded and shrunken (FIG. 20A, panel b). Staining with Hoechst33324 showed increased number of cells with nuclear fragmentation andcondensation (FIG. 20A, panel b). These nuclear changes were abolishedby concurrent treatment with 1 nM SS-31 (FIG. 20A, panel c′). The numberof apoptotic cells was dose-dependently reduced by concurrent treatmentwith SS-31 (P<0.0001) (FIG. 20B).

An increased number of cells with condensed nuclei was also observedwhen SH-SY5Y cells were treated with 25 μM tBHP for 24 h, and the numberof apoptotic cells was dose-dependently reduced by concurrent treatmentwith SS-31 (P<0.0001) (FIG. 20C).

Example 23—SS-31 Protected Against tBHP-Induced Caspase Activation (FIG.21)

Incubation of N₂A cells with 100 JAM tBHP for 24 h resulted in asignificant increase in pan-caspase activity that was dose-dependentlyprevented by co-incubation with SS-31 (P<0.0001 (FIG. 21A). A N₂A cellstreated with 50 μM tBHP for 12 h showed intense staining (red) forcaspase-9 activity (FIG. 21B, panel b). Note that cells that shownuclear condensation all showed caspase-9 staining. Concurrentincubation with 1 nM SS-31 reduced the number of cells showing caspase-9staining (FIG. 21B, panel c).

Example 24—SS-31 Inhibited tBHP-Induced Increase in Intracellular ROS(FIG. 22)

Intracellular ROS production is an early and critical event inoxidant-induced cytotoxicity. Treatment of N₂A cells with 100 μM tBHPresulted in rapid increase in intracellular ROS, as measured by DCFfluorescence, over 4 h at 37° C. (FIG. 22A). Concurrent treatment withSS-31 dose-dependently reduced the rate of ROS production, with 1 nMSS-31 effectively reducing ROS production by >50%. The reduction inintracellular ROS was confirmed by fluorescent microscopy with DCF (FIG.22B). Treatment with N₂A cells with 50 μM tBHP caused significantincrease in DCF fluorescence (green), and this was significantly reducedby co-incubation with 1 nM SS-31 (FIG. 22C).

Example 25—SS-31 Prevented Loss of Mitochondrial Function Caused by tBHP(FIG. 23)

Treatment with low doses of tBHP (50-100 μM) for 24 h resulted in asignificant decrease in mitochondrial function as measured by the MTTassay in both cell lines. Only viable mitochondria containing NADPHdehydrogenase activity are capable of cleaving MTT to the formazan. A 50μM tBHP induced 50% loss of mitochondrial function in N₂A cells (FIG.23A, P<-0.01) and 30% loss of mitochondrial function in SH-SY5Y cells(FIG. 23B, P<0.01). Concurrent treatment with SS-31 dose-dependentlyreduced tBHP-induced mitochondrial toxicity in both N₂A (FIG. 23A;P<0.0001) and SHSY5Y cells (FIG. 23B; P<0.0001). The non-scavengingpeptide, SS-20, did not protect against tBHP-induced mitochondrialdysfunction in N₂A cells (FIG. 23A). Treatment of N₂A cells with SS-31alone had no effect on mitochondrial function.

Example 26—Increased Hydrogen Peroxide (H₂O₂) Sensitivity of G93A-SODTransfected Murine Neuroblastoma (N2a) Cells (FIG. 24)

In a cell culture model of N₂A cells overexpressing either wild type ormutant (69A-SOD1, the mutant cells were significantly more sensitive toH₂O₂-induced cell death both at 0.04 mM and 1 mM concentrations. Thiscell-death was significantly reduced by addition of SS-31 inconcentration between 1 and 100 μM to the medium 1 h after exposure toH₂O₂(FIG. 24).

Example 27—SS-31 Increased Survival of G93A Transgenic Familial ALS Mice(FIG. 25)

Treatment with 5 mg-kg SS-31 i.p. started at 30 days of age of G93Atransgenic familial amyotrophic lateral sclerosis (ALS) mice (high copynumber, B6SJL-Tg(SOD1-G93A)1Gur/J) led to a significant delay of diseaseonset as defined by the appearance of tremor and hind limb clasping aswell as deterioration in the rotarod performance, the average age ofonset in the control group was 88+7 days, in the SS-31 treated group95+6 days (p<0.05, Logrank (Mantel-Cox)). Survival was significantlyincreased by SS-31 treatment from 130±12 to 142+12 day (i.e., 9%)(p<0.05, Logrank (Mantel-Cox)) (FIG. 25). Treatment was well toleratedand no side effects were observed. There was a gender effect on survivalwhich has been observed in previous studies with this model as well inG93A mice in this background with males having a shorter life span thanfemales (average of 5 days). This gender difference was seen in bothgroups and not modified by the treatment.

Example 28—Effect of SS-31 on Motor Performance of G93A TransgenicFamilial ALS Mice (FIG. 26)

Motor performance was significantly improved in the SS-31 treated micebetween day 100 and 130 (p<0005, Repeated measures ANOVA followed byFisher's PLSD) (FIG. 26).

Example 29—Attenuation of Motor Neuron Loss by SS-31 in the Ventral Hornof the Lumbar Spinal Cord of G93A Mice. (FIGS. 27.28 and 29)

Stereological cell counts in the lumbar spinal cord revealed asignificant cell loss in the vehicle treated G93A mice as compared tonon-transgenic littermate control animals. The cell loss wassignificantly ameliorated by administration of SS-31 (FIG. 27).Immunostaining for markers of oxidative and nitrosative stress(4-hydroxynonenenal, 3-nitrotyrosine) showed increased levels of lipidperoxidation and protein nitration in the spinal cord of G93A ascompared to control mice. This was markedly reduced in the SS-31 treatedmice (FIG. 28, 4-hydroxynonenenal; FIG. 29, 3-nitrotyrosine).

Example 30—Reduced Ascorbate and GSH Levels in Post-Ischemic Brain (FIG.30)

Ascorbate and GSH, in addition to cysteine were determined in thepostischemic cortex and striatum. While cysteine levels were generallydecreased in both hemispheres after ischemia, they were significantlyhigher in the ipsilateral side compared to the contralateral side (FIGS.30A and B). By contrast, the levels of ascorbate and GSH, the majorwater-soluble intracellular antioxidants in brain, were progressivelydecreased in the ipsilateral side within a few hours of reperfusion(FIGS. 30C-F). Antioxidant reduction was significant in both cortex andstriatum at 6 h and was further reduced at 24 h, at a time when theinfarct is visible.

Example 31—Treatment with SS-31. But Not SS-20. Attenuates GSH Depletionin Post-Ischemic Cortex (FIG. 31)

To test the efficacy of SS-3 ion redox status, cysteine, ascorbate andGSH levels were determined 6 h after 30 min transient middle cerebralartery occlusion (MCAO) in mice treated intraperitoneally (i.p.) withsaline (vehicle), SS-31 (2 mg/kg) or SS-20 (2 mg/kg) upon reperfusion.Values in FIG. 31 are expressed as percent increase (cysteine) andpercent depletion (ascorbate and GSH) in the ipsilateral side comparedto the contralateral side. Absolute values were originally expressed asnmoles/mg protein as shown in FIG. 30 and converted to percentdifference. The percent ipsilateral cysteine increase was similar amongvehicle-, SS-31-, and SS-20-treated groups (FIG. 31A). Percentipsilateral depletion of ascorbate was marginally but not significantlyaffected in both SS-31 and SS-20-treated animals (FIG. 31B). Incontrast, ischemia-induced GSH depletion in the cortex was significantlyattenuated in SS-31-treated animals compared to the vehicle-treatedgroup (FIG. 31C).

The degree of ipsilateral GSH depletion in SS-20-treated mice was notsignificantly different from that of vehicle-treated mice (FIG. 31C).The data show that SS-31 assists in maintaining antioxidant status andprotects against ischemia-induced depletion of GSH in the cortex.

Example 32—Treatment with SS-31 Peptide Reduces Infarct Size andSwelling (FIG. 32)

To address whether SS-31-induced attenuation in GSH depletion isassociated with neuroprotection, mice were subjected to 30 or 20 minMCAO and then treated with vehicle or SS-31 (2 mg/kg, i.p.) uponreperfusion) and at 6 h, 24 h, 48 h after MCAO. Infarct volumes andhemispheric swelling were determined at 3 days after ischemia. SS-31treatment resulted in moderate but significant reduction in infarctvolume following both 30 min (24% reduction) and 20 min (30% reduction)ischemia, (FIGS. 32A-C). Treatment with SS-31 also significantlyattenuated hemispheric swelling in both 30 and 20 min ischemic paradigms(FIG. 32D). There was no difference in cerebral blood flow (CBF)reduction during MCAO (FIG. 32E) and reperfusion at 10 min (FIG. 32F)between vehicle- and SS-31-treated groups, indicating that theneuroprotective effect by SS-31 occurs via mechanisms other than alteredCBF during and after the ischemic insult.

Example 33—Islet Cell Uptake of SS-31 (FIG. 33)

Islets are tightly adherent cell clusters and entry of peptides/proteinsmay be impaired given their architecture. FIG. 33A shows that SS-31readily penetrates intact mouse islets; in four consecutive experiments,the mean (±SE) of [³H]SS-31 uptake was 70.2±10.3 pmol/mg of protein.

Example 34—SS-31 Prevents Mitochondrial Depolarization (FIG. 33)

Mitochondrial depolarization and the release of cytochrome c into thecytoplasm are critical antecedent events to cell death. TMRM, afluorescent cationic indicator is taken up into mitochondria in apotential dependent manner. FIG. 33B confocal laser scanning microscopicimaging of TMRM treated islets shows that the islets from mice treatedwith SS-31 exhibit greater uptake of TMRM compared to islets from micenot treated with SS-31.

Example 35—Optimization of Islet Isolation with SS-31

To investigate whether the SS-31 optimizes islet isolation and resultsin increased islet yield, islet donor mice were pre-treated with SS-31.SS-31 resulted in a significantly higher islet cell yield compared tountreated mice the mean±(SE) islet yield from the pancreata harvestedfrom SS-31 pre-treated mice was 291±60 islets per pancreas (N=6 separateislet isolations from 28 pancreata) compared to 242±53 islets perpancreas retrieved from the control mice (N=6 separate islet isolationsfrom 30 pancreata) (P=0.03, Two-tailed Pvalue calculated withMann-Whitney test).

Example 36—SS-31 Reduces Islet Cell Apoptosis (FIG. 34)

SS-31 treatment, in addition to enhancing islet yield resulted in asignificant decrease in early as well as late, islet cell apoptosis.Dual parameter flow cytometry analyses of islet cells stained with bothAnnexin V and propidium iodide demonstrated that the treatment of isletdonor mice with SS-31 reduced the percentage of early apoptotic cells(Annexin V alone positive cells) from 11.4±2.4% to 5.5±1.0% (FIG. 34A,P=0.03). SS-31 treatment reduced late apoptosis/early necrosis (AnnexinV+/PI+cells) from 22.7±4.7% to 12.6±1.8 (FIG. 34B, P=0.03) and increasedislet cell viability (Annexin V−/PI-cells) from 47±5.1% to 62±3.5% (FIG.34D, P=0.03)). SS-31 treatment of islet donor mice, however, did notreduce the percentage of necrotic cells (PI+cells: 20±3.2% vs. 19±4.7%,P=1.0).

Example 37—SS-31 Improves Post-Transplant Islet Graft Function (FIG. 35)

The SS-31 treatment associated decrease in islet cell apoptosis andenhanced viability had a beneficial functional consequence. In amarginal islet cell mass transplantation model, 0 of 8 recipients ofislets isolated from pancreas harvested from the control mice hadsuccessful reversal of hyperglycemia (defined as three consecutive bloodglucose levels<200 mg/dl), whereas sustained normoglycemia occurred in 5of ten recipients of islets isolated from pancreas from the SS-31treated donor mice (FIG. 35). It is worth noting that reversal ofhyperglycemia was prompt (by day 1 post-transplant) and discontinuationSS-31 treatment of the islet graft recipient on day 10 did not result inreturn of hyperglycemia demonstrating a sustained effect of SS-31treatment on islet cell function.

What is claimed is:
 1. A method for preventing loss ofdopamine-producing neurons in a mammal having or suspected of havingParkinson's disease, the method comprising administering to the mammalan effective amount of a peptide having the formulaD-Arg-Dmt-Lys-Phe-NH₂.
 2. The method according to claim 1, wherein themammal is a human.
 3. The method according to claim 1, wherein thepeptide is administered orally, topically, intranasally, systemically,intravenously, subcutaneously, intramuscularly,intracerebroventricularly, intrathecally, or transdermally.
 4. Themethod of claim 1, wherein the peptide is mixed with a pharmaceuticallyacceptable carrier.
 5. A method for treating amyotrophic lateralsclerosis (ALS) in a mammal, the method comprising administering to themammal an effective amount of a peptide having the formulaD-Arg-Dmt-Lys-Phe-NH₂.
 6. The method according to claim 5, wherein themammal is a human.
 7. The method according to claim 5, wherein thepeptide is administered orally, topically, intranasally, systemically,intravenously, subcutaneously, intramuscularly,intracerebroventricularly, intrathecally, or transdermally.
 8. Themethod of claim 5, wherein the peptide is mixed with a pharmaceuticallyacceptable carrier.